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THE  WASSERMANN  TEST 


THE  WASSERMANN  TES 


BY 

CHAELES  F.  CRAIG,  A.M.  (Hon.),  M.D.  (Yale) 

LIEUTENANT   COLONEL,   MEDICAL   CORPS,    UNITED    STATES   ARMY;    FELLOW    OP   THE 

AMERICAN    COLLEGE    OF    SURGEONS;    FORMERLY   ASSISTANT    PROFESSOR    OF 

BACTERIOLOGY   AND   PATHOLOGY,   ARMY   MEDICAL    SCHOOL,   AND    GEORGE 

WASHINGTON    UNIVERSITY;     COMMANDING    OFFICER,    DEPARTMENT 

LABORATORY,    CENTRAL   DEPARTMENT,    UNITED    STATES    ARMY, 

FORT  LEAVENWORTH,  KANSAS. 


Published  with  authority  of  the  Surgeon  General, 
United  States  Army 


ILLUSTRATED  WITH  COLORED  PLATE8,  HALFTONE 
PLATES,  AND  FIFTY-SEVEN  TABLES 


ST.  LOUIS 

C.  V.  MOSBY  COMPANY 

1918 


Copyright,  1918,  By  C.  V.  Mosby  Company 


Press  of 

C.  V.  Mosby  Company 

St.  Louis 


PEEFACE 

The  following  work  upon  the  Wassermann  test  has  been 
written  at  the  solicitation  of  friends  who  have  urged  me 
to  place  upon  record  the  experience  gained  from  nearly  ten 
years  of  personal  use  of  this  test  in  the  diagnosis  and  as  a 
control  of  the  treatment  of  syphilis.  I  have  tried  to  include 
in  the  work  all  of  the  most  important  data  that  have  been 
published  regarding  this  very  valuable  diagnostic  measure, 
and  the  conclusions  that  I  feel  may  rightly  be  drawn  from 
its  use  in  the  diagnosis,  control  of  treatment,  and  as  an  in- 
dex ol  the  prevalence  of  this  disease. 

The  work  has  been  largely"  prepared  since  the  outbreak 
of  the  present  Avar  and,  for  this  reason,  is  not  as  exhaus- 
tive as  I  had  originally  intended  it  to  be,  as  owing  to  official 
duties  it  has  been  impossible  to  spend  as  much  time  in  its 
preparation  as  would  have  been  necessary  to  make  it  an  ex- 
haustive treatise,  and  it  has  also  been  impossible  for  me 
to  consult  much  of  the  very  extensive  literature  that  has 
accumulated  during  recent  years  in  regard  to  the  test. 
However,  it  is  believed  that  the  work  contains  all  of  the 
essentialiind  really  valuable  facts  regarding  the  test  which 
have  been  reported  in  the  literature,  and  if  there  have  been 
any  omissions  I  would  deem  it  a  favor  to  have  them  called 
to  my  attention. 

I  have  quoted  quite  liberally  from  some  of  the  more  re- 
cent investigators,  as  Noguchi,  Nichols,  Vedder,  and  Kol- 
mer,  and  have  also  used  much  data  previously  published  by 
myself  in  various  medical  journals,  and  it  is  a  pleasure  to 
tender  my  thanks  to  the  editors  of  the  Journal  of  the  Amer- 
ican Medical  Association,  the  Journal  of  Experimental 
Medicine,  the  Journal  of  Infectious  Diseases,  and  the  Amer- 
ican Journal  of  Syphilis,  for  permission  to  avail  myself  of 
the  data  previously  published  in  these  journals. 

11 


12  PREFACE 

From  personal  experience,  I  believe  that  there  is  still 
a  great  deal  of  misunderstanding  and  confusion  among  the 
members  of  the  medical  profession  regarding  the  exact 
value  and  limitations  of  the  Wassermann  test,  both  in  the 
diagnosis  of  s^^philis,  and  when  used  as  a  control  of  the 
treatment  of  the  disease,  and  if  this  work  will  help  in  clear- 
ing np  this  confusion  it  will  be  a  source  of  great  gratifica- 
tion. Much  of  this  misunderstanding  rests  upon  the  shoul- 
ders of  laboratory  workers,  for  it  must  be  admitted  that 
too  often  the  performance  of  the  Wassermann  test  has  been 
delegated  to  poorly  trained  or  careless  assistants,  and  the 
results  obtained  mth  the  test  have  thus  been  erroneous  and 
unsatisfactory.  I  can  not  urge  too  strongly  upon  the  pro- 
fession the  necessity  for  submitting  material  for  this  test 
to  Avell-qualified  serologists  if  reliable  results  are  to  be  ob- 
tained. A  standard  technic  for  the  test  is  much  to  be  de- 
sired but  all  efforts  in  this  direction  have  failed,  omng 
largel}^  to  the  difficulty  of  securing  a  standard  antigen,  so 
that  at  the  present  time  several  methods  of  performing  the 
test  are  in  use,  all  of  which  are  reliable  in  the  hands  of 
experienced  serologists.  The  method  recommended  in  this 
work  has  stood  the  test  of  time  and  has  been  used  by  many 
different  workers  in  thousands  of  syphilitic  infections,  and 
it  is  believed  that  it  is  as  simple  in  technic  and  as  accurate 
in  results  as  any  method  of  performing  the  Wassermann 
test  that  has  been  devised. 

My  thanks  are  due  the  publishers  for  their  courtesy 
and  assistance  in  many  ways  and  for  the  very  excellent 
manner  in  which  they  have  reproduced  the  color  plates  pub- 
lished in  the  work. 

Charles  F.  Craig. 

Department  Laboratory,  Central  Department,  U.  S.  Army, 
Fort  Leavenworth,  Kansas. 
June  1,  1918. 


CONTENTS 

CHAPTER  I 
u)    The  Discovery  of  the  Wassermann  Test 17    ^' 


Q  PAGE 


@ 


CHAPTER  II 

General  Description  of  the  Wassermann   Test.     Nature  of  the 

Reaction 30  u 

CHAPTER  III 

Factors  Which  Influence  the'  Result  of  the  Test 40 

CHAPTER  IV 

Preparation  and  Titration  op  the  Reagents  Used  in  the  Wasser- 
mann Test 60 

CHAPTER  V 

The  Technic  of  the  Writer's  Modification  of  the  Wassermann 
Test.  The  Test  Upon  the  Cerebrospinal  Fluid.  The  Origi- 
nal Wassermann  Technic.  Other  Modifications  of  the 
Wassermann  Test 89 

CHAPTER  VI 

Complement  Fixation  in   Syphilis  with  Antigens  Prepared   prom 

Pure  Cultures  of  Treponema  Pallidum 116 

CHAPTER  VII 

(2)  The  Results  of  the  Wassermann  Test  in  the  Various  Stages  of 
'^  Syphilis.     Time  op  Appearance  of  the  Reaction.     The  Spec-  / 


^ 


ificity  of  the  Wassermann  Test 127 

CHAPTER  VIII 

The  Interpretation  op  the  Results  of  the  Wassermann  Test.  The 
Wassermann  Test  as  an  Index  of  the  Prevalence  of  Syph- 
ilis IN  Communities 152 

13 


14  CONTENTS 

CHAPTER  IX 


The  Effect  of  Treatment  upon  the  Wassermann  Reaction.  The 
Wassermann  Test  as  a  Control  op  the  Treatment  of  Syph- 
Hjs.     The  Provocative  Wassermann  Reaction 171 

CHAPTER  X 

The  Wassermann  Test  upon  the  Cerebrospinal  Fluid.  Interpreta- 
tion op  Results.  The  Colloidal  Gold  Test.  The  Cell 
Count  and  Tests  for  Increase  in  Globulins  in  the  Cerebro- 
spinal  Fluid 205 

BIBLIOGRAPHY 
Bibliography 231 


Q 


ILLUSTRATIONS 

PAGE 

Plate  I.  (1)   Titration  of  complement.     (2)  Titration  of  amboceptor  pa- 
pers.    (3)   Titration  of  antigen 78 

Plate  II.  (1)  Beading  of  results  of  Wassermann  test.     (2)  Positive  reac- 
tion.    (3)   Negative  reaction 98 

Plate  III.  Paretic  reaction.     Tabetic  reaction 218 

FIG. 

1.  Showing  hemolytic  and  bacteriolytic  systems 24 

2.  Schematic  representation   of  complement  fixation  in   syphilis   or  the 

Wassermann  test 32 

3.  Luer   syringes 62 

4.  Electric    centrifuge 66 

5.  Electric  centrifuge  with  shaker  head  attached 70 

6.  Small  incubator 72 

7.  Water-bath  suitable  for  Wassermann  test  incubations 74 

8.  Amboceptor  cutter.     Front  view,  showing  gauge 80 

9.  Amboceptor  cutter.     Rear  view,  showing  •  scale  and  set  screw     ...  82 
10.  Amboceptor  markers 84 


15 


THE  WASSERMANN  TEST 


CHAPTEE  I 
THE  DISCOVERY  OF  THE  WASSERMANN  TEST 

The  discovery  of  the  Wassermann  test  for  the  diagnosis 
of  syphilis  was  the  direct  result  of  the  study  of  inununity 
and  especially  of  the  discovery  of  certain  facts  of  basic 
importance  in  that  process ;  i.  e.,  bacteriolysis,  hemolysis, 
and  complement  fixation. 

Bacteriolysis 

In  1888,  NuttalP  demonstrated  that  freshly  dra\vn  blood 
serum  and  defibrinated  blood  had  a  marked  destructive 
effect  upon  bacteria,  an  effect  that  could  be  definitely  esti- 
mated, and  which  was  undoubtedly  due  to  some  substance 
or  substances  in  the  blood  serum  and  defibrinated  blood. 
This  effect  consisted  in  the  dissolving  of  the  bacteria  and 
the  process  was  called  bacteriolysis,  the  substance  causing 
the  destruction  of  the  bacteria  being  known  as  a  hacter- 
iolysin.  The  bacteriolytic  effect  of  the  blood  serum  or  de- 
fibrinated blood  could  be  demonstrated  in  the  following 
manner : 

Definite  amounts  of  the  blood  serum  or  defibrinated  blood 
were  mixed  with  definite  amounts  of  the  bacterium  experi- 
mented with,  and  incubated  at  37°  C.  for  several  hours, 
after  which  plates  were  poured  and  the  number  of  colonies 
of  bacteria  developing  compared  with  the  number  upon 
plates  containing  the  same  bacterium  but  to  which  no  blood 
serum  or  defibrinated  blood  had  been  added.     It  was  ob- 

17 


18  THE   WASSERMAISTN   TEST 

served  that  the  plates  prepared  from  the  mixtures  of  bac- 
teria and  blood  sermn  and  defibrinated  blood  showed  very 
many  less  colonies  of  the  bacterium  than  those  to  which  no 
blood  serum  or  defibrinated  blood  had  been  added,  and,  in 
some  of  the  experiments,  the  plates  were  almost  sterile. 

Nuttall's  experiments  were  confirmed  by  von  Fodor^  and 
Buchner,^  and  it  was  the  study  and  discussion  of  this  sub- 
ject that  introduced  into  the  science  of  medicine  the  ''hu- 
moral" theories  regarding  the  nature  of  immunity. 

Nuttall  had  shown  that  the  bactericidal  power  of  blood 
serum  was  very  unstable  and  that  it  was  only  present  in 
fresh  blood  or  blood  serum,  and  that  heating  the  serum  at 
55  to  56°  C,  or  allowing  it  to  stand  for  any  considerable 
time  destroyed  its  capacity  for  dissolving  the  bacteria. 
Buchner,*  in  explaining  the  phenomenon  of  bacteriolysis, 
evolved  what  is  now  known  as  the  humoral  explanation, 
i.  e.,  that  the  capacity  of  the  blood  or  serum  for  dissolving 
the  bacteria  resided  in  a  constituent  of  the  fresh  blood 
serum  which  he  termed  ' '  alexin ; ' '  which  he  believed  to  be 
active  against  all  bacteria;  and  which  he  compared  to  the 
action  of  a  ferment.  Buchner  further  stated  that  this 
"alexin"  might  be  of  cellular  origin,  either  a  product  of  the 
tissue  cells  or  leucocytes.  Alexin  is  identical  with  the 
"complement"  of  Ehrlich  and  his  followers,  and  in  this 
work  the  term  "complement"  will  be  used  in  preference 
to  "alexin," 

In  1894,  Pfeiff er,^  by  his  classical  experiment  upon  chol- 
era immunity  in  the  guinea  pig,  demonstrated  that  the 
process  of  bacteriolysis  is  a  specific  one  and  that  the  injec- 
tion of  a  specific  bacterium  into  an  animal  is  followed  by 
the  production  of  a  specific  bacteriolysin  in  the  blood  serum 
of  that  animal  for  the  bacterium  injected.  His  experiment, 
now  a  common  classroom  demonstration  of  bacteriolysis, 
was  briefly  as  follows : 

If  a  guinea  pig,  which  has  recovered  from  cholera,  be  in- 
jected in  the  peritoneal  cavity  with  a  suspension  containing 
cholera  spirilla,  it  will  be  found,  upon  examination  of  the 


DISCOVERY  19 

exudate  from  the  peritoneal  cavity  of  the  animal,  that  the 
spirilla  undergo  rapid  destruction,  all  stages  of  the  process 
being  visible  in  samples  of  the  exudate  taken  at  graduated 
intervals  of  time  after  the  injection.  The  spirilla  will  be 
observed  to  become  sv/ollen,  distorted  in  shape  and  gran- 
ular in  appearance,  and  soon  entirely  disappear,  being  dis- 
solved in  the  peritoneal  exudate.  That  this  process  is  pro- 
tective in  nature  is  shown  by  the  fact  that  such  an  animal 
will  survive  doses  of  the  spirilla  that  are  lethal  to  animals 
that  have  not  been  protected  by  a  previous  attack  of  chol- 
era, and  that  it  is  specific  by  the  fact  that  other  bacteria, 
when  injected  into  the  peritoneal  cavity  of  the  cholera  pig, 
are  unaffected. 

In  other  words,  the  experiment  proved  that  the  pig,  after 
recovery  from  cholera,  had  present  in  the  peritoneal  ex- 
udate some  protective  substance,  specific  in  nature,  which 
was  capable  of  dissolving  the  cholera  spirilla  and  incapable 
of  acting  upon  other  bacteria.  This  substance  was  the 
same  as  that  demonstrated  by  Nuttall  in  fresh  blood  serum 
and  defibrinated  blood  and  to  which  the  name  ''bacterio- 
lysin"  had  been  given. 

A  very  significant  phenomenon  observed  by  Pfeiffer  was 
that  if  the  peritoneal  cavity  of  a  normal  guinea  pig  was 
injected  with  a  mixture  of  cholera  spirilla  and  some  blood 
serum  from  the  immunized  pig,  the  normal  pig  also  escaped 
infection.  That  is,  it  was  possible  thus  to  transfer  the  bac- 
teriolytic power  possessed  by  the  peritoneal  exudate  of  the 
immunized  pig  to  the  peritoneal  cavity  of  an  unprotected 
pig  by  mixing  the  cholera  spirilla  with  the  blood  serum  of 
the  protected  animal,  thus  proving  that  the  bacteriolysin 
also  existed  in  the  blood  serum.  It  was  also  found  that 
while  the  bactericidal  power  of  the  blood  serum  of  the  im- 
munized animal  was  destroyed,  in  vitro,  by  heating,  the 
heated  blood  serum  is  just  as  effectual  in  producing  im- 
munity in  a  normal  guinea  pig  when  injected  with  the  chol- 
era spirilla  as  the  unheated.  This  fact  Pfeitfer  thought 
to  be  due  to  the  necessity  of  some  living  tissue  taking  part 


20  THE   WASSERMAlSri^   TEST 

in  the  reaction  and  stated  that  the  endothelial  cells  of  the 
peritoneum  were  probably  the  cells  concerned  in  this  par- 
ticular exj)erinient.  His  conclusions,  in  this  respect,  lead 
him  to  believe  that  bacteriolysis,  so  far  as  it  occurred  with 
the  cholera  spirillum,  could  only  take  place  in  the  living 
body. 

The  researches  of  Pfeiffer  stimulated  a  great  deal  of  re- 
search upon  the  subject  and  to  Bordet  we  owe 'the  true  ex- 
planation of  the  process  of  bacterioh'sis.  Pfeiffer's  con- 
ception that  it  could  only  occur  in  the  living  animal  was 
disproved  by  Bordet®  who  produced  bacteriolysis  of  the 
cholera  spirillum  in  hanging  drop  preparations,  and 
showed,  further,  that  while  the  bacteriolytic  action  of  im- 
mune serum  is  destroyed  by  heating  the  serum  at  56°  C, 
the  addition  to  this  so-called  ''inactivated"  serum  of  a 
small  amount  of  fresh  normal  blood  serum,  will  "reac- 
tivate" or  restore  the  bacteriolytic  property  of  the  serum. 

Thus,  while  normal  blood  serum  may  contain  no  bacte- 
riolytic substances  against  the  organism  experimented 
with,  when  added  to  an  innnune  serum  which  has  been 
heated  and  its  bacteriolytic  activity  destroyed,  it  has  the 
power  of  again  bringing  the  bacteriolytic  properties  of 
the  immune  serum  into  play  and  bacteriolysis  results. 
From  this  experiment  it  is  evident  that  in  the  unheated 
innnune  serum  the  dissolving  of  the  bacterium  must  de- 
pend upon  tivo  substances,  one  rendered  powerless  by  heat, 
while  the  other  is  unaffected.  The  substance  rendered  in- 
active by  heat  is  said  to  be  tliermolahile,  while  the  unaf- 
fected substance  is  said  to  be  tliermo stable.  It  is  also  evi- 
dent that  the  one  can  not  act  without  the  other  upon  the 
bacterium,  and  that  the  thermolabile  body  is  present  in 
normal,  unheated  serum,  while  the  thermostable  body  is 
only  present  in  the  immune  serum.  Bordet 's  researches 
also  proved  the  specific  nature  of  the  bacteriolysins  and 
that  an  animal  immunized  to  several  specific  bacteria  will 
produce  in  its  blood  specific  bacteriolysins  against  the  va- 
rious bacteria  injected  during  the  process  of  immunization. 


DISCOVEEY  21 

From  the  researches  mentioned  it  is  evident  that  the 
process  of  bacteriolysis  depends  upon  the  production  in 
the  hlood  of  an  animal  of  a  specific  protective  substance 
ivhich,  in  the  presence  of  fresh  normal  blood  serum,  is  ca- 
pable of  dissolving  the  specific  bacterium  against  ivhich 
the  animal  has  been  immunized.  For  bacteriolysis  to  occur 
there  must  be  present  three  factors;  one,  the  bacterium 
used  in  immunisation;  the  second,  the  blood  serum  contain- 
ing the  protective  substance  produced  by  immunization; 
and  third,  the  substance  ivhich  is  present  in  fresh  normal 
blood  serum. 

In  immunological  literature  certain  terms  have  been 
given  the  factors  mentioned  and  in  this  work  those  used  by 
Ehrlich  and  his  followers  will  be  used.  Thus,  the  bac- 
terium injected  in  order  to  produce  the  bacteriolytic  sub- 
stance is  called  an  antigen,  and  this  name  may  be  applied  to 
any  substance,  which  when  injected  into  an  animal,  results 
in  the  formation  of  an  antibody.  The  bacteriolytic  sub- 
stance, itself,  is  called  an  antibody  or  amboceptor ;  while 
the  substance  which  is  present  in  fresh  normal  blood  serum 
and  which,  when  brought  into  contact  with  the  antigen  and 
amboceptor,  causes  bacteriolysis  is  called  complement. 
This  substance  is  identical  with  Buchner's  '^alexin." 

Definitions. — As  these  terms  are  used,  throughout  this 
work,  in  describing  the  Wassermann  reaction,  the  follow- 
ing definitions  of  them  are  inserted  for  convenience,  as  it 
is  essential  that  they  be  understood  if  one  desires  to  have 
a  clear  conception  of  the  test : 

ANTiGE:sr. — Any  substance  which,  when  injected  into  suit- 
able animals,  will  result  in  the  formation  of  specific  anti- 
bodies. Antigens  may  be  bacterial,  cellular,  or  chemical  in 
nature. 

Amboceptor. — The  specific  antibody  produced  by  the  in- 
jection of  an  antigen.  Amboceptors  may  be  normally  pres- 
ent in  certain  blood  sera  but  can  be  greatly  increased  by 
the  injection  of  the  suitable  antigen.  Amboceptors  are 
thermostable,    retaining    their    activity    after    heating    at 


22  THE   WASSERMANE"   TEST 

56°  C.  for  one  half  hour.  A  hacteriolysin  is  a  bacterio- 
lytic amboceptor.    A  hemolysin  is  a  hemolytic  amboceptor. 

Complement. — The  substance  present  in  all  fresh  nor- 
mal sera,  which  when  added  to  a  mixture  containing  anti- 
gen and  amboceptor,  results  in  the  production  of  bacterio- 
lysis or  hemolysis.  Complement  is  rendered  inactive  by 
heating  for  one  half  hour  at  56°  C.  and  is,  therefore,  ther- 
molabile.  It  also  loses  strength  rapidly  on  standing  at 
room  temperature. 

Bacteriolytic  System. — The  combination  of  antigen 
(bacterium),  amboceptor  (immune  serum),  and  comple- 
ment (normal  serum)  is  called  a  bacteriolytic  system. 

Hemolysis 

During  his  researches  upon  the  nature  of  bacteriolysis 
Bordef  discovered  that  if  an  animal  be  injected  with  the 
red  blood  corpuscles  of  another  animal  a  substance  is  pro- 
duced in  the  blood  serum  of  the  injected  animal  capable  of 
laking  the  red  corpuscles  injected.  To  this  substance  the 
name  'Hiemolysin''  is  generally  applied  and  the  process  of 
laking  is  kno^Ti  as  hemolysis.  Bordet's  discovery  of  hem- 
olysis was  announced  in  1898  and  at  the  same  time  Belfanti 
and  Carbone^  also  described  their  experiments  demonstrat- 
ing the  same  phenomenon,  so  that  to  these  investigators 
belongs  equally  the  credit  for  the  discovery  of  one  of  the 
most  useful  and  important  phenomena  of  immunity.  Al- 
though foreshadowed  by  the  work  of  Landois,  who,  in  1875, 
obsei^ed  that  the  blood  serum  of  certain  animals  produced 
laking  of  the  blood  of  other  species  when  the  two  were 
added  together,  the  real  explanation  of  the  process  was 
made  clear  by  the  Avork  of  Bordet,  who  proved  that,  while 
hemolysins  are  sometimes  present  in  the  freshly  drawn 
blood  of  certain  species  against  the  cells  of  other  species 
of  animals,  they  can  be  produced  in  large  amount  by  the 
injection  of  the  washed  erythrocytes  of  one  species  into 
another,  and  that  the  hemolysins  thus  produced  are  abso- 
lutely specific  in  nature. 


DISCOVERY  23 

Thus,  if  a  rabbit  be  injected  with  the  washed  red  blood 
corpuscles  of  man,  the  hemolysin  thus  produced  in  the 
rabbit's  blood  serum  will  cause  laking  of  human  erythro- 
cytes but  will  be  powerless  against  erythrocytes  of  other 
species;  if  injected  with  the  erythrocytes  of  a  sheep,  the 
hemolysin  produced  will  only  act  upon  sheep's  erythro- 
cytes ;  if  injected  with  the  erythrocytes  of  an  ox,  the  rab- 
bit's blood  serum  will  only  hemolyse  ox  corpuscles.  In 
other  words,  hemolysins  are  absolutely  specific  against  the 
kind  of  erythrocytes  injected  in  immunizing  an  animal  and 
are  powerless  against  other  erythrocytes. 

As  will  be  seen,  the  hemolysins  are  strictly  analogous  to 
the  bacteriolysins,  being  produced  by  the  injection  of  an 
antigen,  the  erythrocytes,  the  only  difference  being  that 
in  the  one  case  bacteria  are  dissolved  while  in  the  other  the 
hemoglobin  is  dissolved  from  the  red  blood  corpuscles  em- 
ployed during  immunization.  In  both  instances  the  reac- 
tion will  not  occur  except  in  the  presence  of  complement, 
which  is  furnished  by  the  addition  of  fresh  normal  blood 
serum,  and  in  both  instances  heating  for  one  half  hour  at 
56°  C.  destroys  the  activity  of  the  immune  serum,  as  it  de- 
stroys the  complement  present  in  the  serum.  Natural  hem- 
olysins occur  in  the  blood  of  one  species  of  animal  against 
the  erythrocytes  of  another,  to  a  limited  extent,  just  as  nat- 
ural bacteriolysins  against  certain  bacteria  occur  in  the 
blood  of  certain  animals,  but  these  may  be  greatly  in- 
creased in  amount  by  the  injection  of  suitable  erythrocytes. 

To  all  bacteriolysins  and  hemolysins  the  group  term 
"cytolysins"  has  been  applied,  owing  to  the  fact  that  their 
injurious  action  is  upon  the  cells  injected  as  antigen  in 
immunization.  In  the  case  of  the  bacteriolysins  the  bacte- 
rium injected  constitutes  the  cell  used  as  antigen,  and  the 
injection  is  folloAved  by  the  production  in  the  blood  serum 
of  a  bacteriolysin  capable  of  destroying  the  bacterium  in- 
jected ;  while  in  the  case  of  the  hemolysins,  the  erythrocyte 
injected  constitutes  the  antigen  and  is  followed  by  the  pro- 
duction in  the  animal's  blood  serum  of  hemolysins  capable 


24  THE    WASSERMANN    TEST 

of  laking  the  erythrocytes  of  the  species  of  animal  from 
which  the  injected  erythrocytes  were  obtained. 

Hemolytic  System. — The  combination  of  antigen  (ery- 
throcytes), amboceptor  (immune  serum)  and  complement 
(normal  serum)  is  called  a  hemolytic  system. 

The  various  theories  that  have  been  advanced  in  expla- 
nation of  the  cause  of  bacteriolysis  and  hemolysis  and  the 
relative  part  played  in  the  process  by  antigen,  amboceptor, 
and  complement,  will  not  be  discussed  here,  as  it  is  unnec- 
essary in  the  consideration  of  the  Wassermann  test. 

Complement  Fixation 

In  1906,  Bordet  and  Gengou^  published  their  paper  de- 
scribing complement  fixation  and  as  the  Wassermann  test 

Hemolytic   system.  Bacteriolytic    system 


B 


0^233 


I    .  II 

Fig.  1. 
I. — A.  Hemolytic  system;  a,  red  blood  corpuscle,  or  antigen;  b,  immune  rabbit  serum,  or 
amboceptor;   c,   guinea  pig  serum,   or  complement. 
B.   Showing  union  of   antigen,   a;  amboceptor,    b;  and  complement,    c,   with   resulting 
hemolysis. 
II. — A.  Bacteriolytic  system;   a,   bacterium   or  antigen;   b,  immune   rabbit  serum,    or  ambo- 
ceptor;  c,  guinea  pig  serum,  or  complement. 
B.   Showing  union   of  antigen,   a;  amboceptor,    b;   and   complement,    c,   with   resulting 
bacteriolysis. 

is  based  upon  this  reaction  it  is  essential  that  it  be  thor- 
oughly understood  before  the  technic  of  the  test,  can  be 
considered. 

In  experiments  upon  bacteriolysis  and  hemolysis  it  will 
be  remembered  that  neither  process  can  occur  without  the 
presence  of  three  factors ;  i.  e.,  antigen,  amboceptor,  and 


DISCOVERY  25 

complement.  For  instance,  if  the  red  corpuscles  of 
a  sheep  be  added  to  the  blood  senim  of  a  rabbit  which  has 
been  immunized  to  these  corpuscles,  nothing  occurs,  but  if 
to  this  mixture  a  little  fr-esh  normal  blood  serum  be  added, 
the  hemoglobin  of  the  red  cells  is  dissolved  from  them  and 
hemolysis  occurs ;  in  the  same  Avay,  if  the  erythrocytes  and 
complement,  or  fresh  normal  blood  serum,  be  placed  to- 
gether, nothing  will  occur  until  the  immune  serum,  or  am- 
boceptor be  added,  when  hemolysis  results.  Furthermore, 
in  neither  case  is  the  complement  in  the  normal  blood  se- 
rum fixed  by  either  antigen  or  amboceptor  alone,  but  can 
only  be  fixed,  or  used  up,  when  both  antigen  and  ambocep- 
tor are  combined  with  it.  In  the  reaction  the  amboceptor, 
contained  in  the  immune  serum,  apparently  acts  as  a  con- 
necting link,  uniting  the  antigen  and  the  complement  and 
making  hemolysis  possible. 

This  fact  led  Bordet  and  Gengou  to  the  discovery  of  com- 
plement.,fixation  as  a  specific  test  for  the  differentiation  of 
bacteria,  for  they  found  that  by  using  a  bacteriolytic  and 
hemolytic  system  together  it  is  possible  to  discover  the 
presence  of  a  specific  antigen  or  specific  amboceptor  in  a 
sjnixture.  In  complement-fixation  tests  the  hemolytic  sys- 
tem is  used  as  an  indicator,  as  hemolysis  is  visible  to  the 
unaided  eye,  while  bacteriolysis  is  not. 

In  order  that  complement  fixation,  as  applied  to  the  dem- 
onstration of  specific  immune  bodies  in  the  blood  serum 
may  be  understood  by  the  reader,  the  following  hypothet- 
ical experiment  will  be  described.  The  complement-fixa- 
tion test  is  not  used  in  the  diagnosis  of  typhoid  fever  prac- 
tically, because  it  is  complicated  and  the  agglutination  test 
for  the  disease  is  more  practical  and  easier  of  application, 
but  the  experiment  described  can  be  carried  out  in  the  prac- 
tice if  desired. 

Problem:  A  patient  is  supposed  to  be  suffering  from  typhoid  fever  and  it 
is  desired  to  determine  the  diagnosis,  if  possible,  by  means  of  complement 
fixation. 

Procedure. — In  a  test  tube  containing  the  necessary  amount  of  normal  salt 


26  '  THE   WASSEEMANN   TEST 

solution  there  is  placed  a  certain  amount  of  the  patient 's  serum,  which  has  been 
inactivated  by  heating  at  56°  C.  for  one  half  hour;  some  normal  guinea  pig 
serum  or  complement,  and  a  certain  amount  of  a  suspension  of  typhoid  bacilli, 
or  antigen.  This  mixture  is  allowed  to  stand  in  a  water-bath  at  37°  C.  for 
one-half  hour,  after  which  there  are  added  a  certain  amount  of  a  suspension 
of  human  red  blood  corpuscles  and  some  serum  from  a  rabbit  which  has  been 
immunized  against  these  erythrocytes.  This  mixture  is  allowed  to  stand  in 
the  water-bath  at  37°  C.  for  one  hour,  placed  in  the  ice  box  for  one  hour, 
and  the  results  then  noted. 

Eesult. — If  it  is  remembered  that  complement  is  absolutely  necessary 
in  order  that  hemolysis  occur  and  that  complement  can  not  be  fixed  or  used 
up  unless  both  antigen  and  amboceptor  be  present  with  it,  the  result  of 
this  experiment  can  be  easily  understood.  If  the  patient  is  really  suffering 
from  typhoid  fever  and  immune  substances  or  amboceptor  be  present  in  the 
blood  serum,  the  complement  in  the  fresh  guinea  pig  serum  which  is  added 
to  the  typhoid  bacillus  suspension,  or  antigen,  and  the  patient's  serum  which 
contains  amboceptor  against  the  typhoid  bacillus,  will  be  used  up  or  fixed 
and  bacteriolysis  will  occur.  If  now  the  erythrocytes  and  the  rabbit's  serum 
containing  amboceptor  agVinst  them  be  added  to  the  mixture,  hemolysis  will 
not  occur,  because  the  complement  has  already  been  used  up  or  fixed  by  the 
bacteriolytic  system  and  is  no  longer  available  for  union  with  the  hemolytic 
antigen  and  amboceptor.  The  test  would,  therefore,  be  positive  for  typhoid, 
as  it  would  demonstrate  that  the  patient's  blood  serum  contained  amboceptor 
or  immune  bodies  for  the  typhoid  bacillus.  On  the  other  hand,  if  the  patient 
did  not  have  typhoid  the  complement  would  not  be  fixed,  so  that  when  the 
erythrocytes  and  immune  rabbit  serum  were  added,  hemolysis  would  occur, 
and  the  test  would  be  called  a  negative  one. 

From  this  experiment  it  will  be  seen  that  the  principle  of 
complement  fixation  can  be  utilized  in  the  diagnosis  of  dis- 
eases dne  to  a  specific  microorganism  and  this  fact  was 
quickly  recognized  and  investigators  endeavored  to  work 
out  complement-fixation  tests  for  many  of  the  acute  spe- 
cific infections.  Since  the  discovery  of  complement  fixation 
the  test  has  been  applied  to  pertussis,  gonorrheal  infec- 
tions, tuberculosis,  glanders,  certain  helminthic  infections, 
and  several  other  conditions,  but  its  greatest  practical  im- 
portance was  reached  in  the  discovery  by  Wassermann, 
Neisser  and  Bruck  of  the  complement-fixation  test  for 
syphilis,  or,  as  it  is  commonly  known,  the  "Wassermann  re- 
action. 

Discovery  of  the  Wassermann  Complement-Fixation  Test 
for  Syphilis. — Many  students  of  the  subject,  after  the  dis- 


DISCOVERY  27 

covery  of  complement  fixation  by  Bordet  and  Gengou,  were 
able  to  show  that  it  was  not  necessary  to  employ  the  whole 
bacteria  as  antigen  in  making  tests  but  that  watery  extracts 
of  the  bacteria,  when  used  as  antigen,  gave  equally  good 
results.    This  fact  caused  Wassermann  and  Bruck  to  con- 
clude that  tuberculins  should  act  well  as  antigen  in  a  com- 
plement-fixation   test    for  tuberculosis,  and  it  was  while  i 
working    upon  this  problem  that  Wassermann    conceived  | 
the  idea  that  complement  fixation  could  be  applied  to  the  i 
diagnosis  of  syphilis.    The  discovery  of  Treponema  palli- 
dum, by  Schaudinn,  had  shown  that  the  disease  was  due  to 
an  organism  that  is  present  in  large  numbers  in  the  tissues 
of  certain  organs,  and  to  these  organs  Wassermann  turned 
for  his  antigenic  extract,  as  Treponema  pallidum  had  not 
then  been  grown  in  pure  culture. 

Wassermann,  working  in  conjunction  with  Neisser  and  Sxpz, 
Bruck,^°  attempted  to  produce  complement  fixation  in  syph-  \ 
ilis  by  using  the  blood  serum  of  syphilitic  monkeys  as  am- 
boceptor and  watery  extracts  of  syphilitic  organs  as  anti- 
gen, and,  as  expected,  it  was  found  that  such  a  combination 
resulted  in  complement  fixation.  It  was  but  a  step  from 
this  experiment  to  the  application  of  the  test  to  man,  and 
in  1906,  Wassermann,  Neisser,  Bruck,  and  Schucht"  pub- 
lished a  paper  giving  their  results  in  the  diagnosis  of  syph- 
ilis in  man  by  the  complement-fixation  test.  Their  paper 
was  antedated  a  short  time  by  that  of  Detre,^^  but  as  Was- 
sermann, Neisser  and  Bruck,  in  their  previous  publication 
describing  the  test  upon  syphilitic  monkeys  antedated 
Detre's  paper,  the  credit  for  the  discovery  remains  with 
Wassermann  and  his  colleagues. 

Wassermann  considered  the  complement-fixation  reac- 
tion in  syphilis  as  a  true  antigen-antibody  reaction,  the 
fixation  being  due  to  specific  antibodies  or  amboceptors 
present  in  the  patient's  blood,  produced  by  Treponema  pal- 
lidum, the  etiological  agent  of  the  disease.  This  conception 
of  the  reaction  was  held  by  all  observers  until  1907,  when 
several  investigators  demonstrated  that  complement  fixa- 


Z6  THE   WASSERMANN   TEST 

tion  in  syphilis  occurred  not  only  when  an  extract  of  tissues 
containing  Treponema  'pallidum  Avas  used  as  antigen,  but 
also  when  nonspecific  substances  were  used,  as  extracts  of 
normal  tissues  and  even  certain  chemical  reagents.  In 
1907,  Weygandt^^  obtained  a  positive  reaction  in  tabes  by 
using  an  antigen  obtained  by  extracting  a  normal  spleen, 
and  Marie  and  Levaditi"  obtained  positive  reactions  in 
paresis  with  an  aqueous  extract  of  normal  liver.  Later, 
Plaut,^^  Landsteiner,"  and  others  obtained  positive  results 
in  complement  fixation  in  syphilis  by  using  aqueous  ex- 
tracts of  normal  organs,  both  of  man  and  animals,  as  anti- 
gen, and  finally,  the  publication  of  the  paper  by  Land- 
steiner,  Miiller  and  Potzl,"  in  which  they  demonstrated 
that  as  good  results  could  be  obtained  in  the  diagnosis  of 
syphilis  by  the  complement-fixation  test  with  alcoholic  ex- 
tracts of  guinea  pig  heart  as  antigen  as  when  aqueous  ex- 
tracts of  foetal  syphilitic  liver  were  employed,  demon- 
strated that  the  reaction  could  no  longer  be  considered  spe- 
cific in  the  sense  that  it  is  a  true  antigen-antibody  reaction. 
The  work  of  the  last  named  investigators  was  soon  con- 
firmed by  others  and  it  was  soon  shown  that  alcoholic  ex- 
tracts of  tissues  were  as  valuable  as  antigens  in  the  Was- 
sermann  test  as  waterj^  extracts,  and  that  normal  organs 
of  man  or  animals  were  apparently  as  rich  in  complement- 
fixing  substances  as  Avere  organs  containing  Treponema 
pallidum. 

Noguchi^*  added  greatly  to  our  knoAvledge  of  the  subject 
by  proving  that  the  substances  which  fix  complement  in  the 
Wassermann  reaction  are  acetone  insoluble,  thus  indicating 
that  they  consisted  largely  of  lipoids,  and  a  further  anal- 
ysis showed  that  such  antigenic  extracts  contained  lecithin, 
cholesterin,  and  sodium  oleate.  The  fact  that  the  addition 
of  a  certain  amount  of  cholesterin  to  antigens  used  in  the 
diagnosis  of  syphilis  greatly  increased  their  antigenic 
value  Avas  first  noted  by  BroAvning  and  Cruickshank^^  and 
was  soon  confirmed  by  other  Avorkers,  notably,  in  this  coun- 


DISCOVERY  29 

try,  by  Walker  and  Swift,-"  and  at  the  present  time  such 
cholesterinized  antigens  are  very  generally  used  and,  in 
the  experience  of  the  writer  and  most  authorities  who  have 
used  them,  are  perfectly  reliable  when  properly  controlled 
and  are  more  valuable  in  the  specific  diagnosis  of  syphilis 
by  the  Wassermann  test  than  are  antigens  which  have  not 
been  thus  fortified. 

The  discovery  that  the  antigens  used  in  the  Wassermann 
test  need  not  be  prepared  from  tissues  containing  Trepo- 
7iema  pallidum  but  that  alcoholic  extracts  of  normal  tissues 
are  just  as  effective,  and  that  certain  chemicals,  as  choles- 
terin,  when  added  to  antigens  prepared  from  normal  tis- 
sues, increase  their  antigenic  value  in  the  diagnosis  of  the 
disease,  demonstrated,  beyond  question,  that  the  early  con- 
ception of  Wassermann  that  complement  fixation  in  syph- 
ilis is  a  specific  antigen-antibody  reaction,  is  erroneous,  and 
that  this  reaction  can  no  longer  be  considered  a  specific 
one  on  the  strict  sense  in  which  this  term  is  employed  in 
immunology.  Although  this  is  undoubtedly  true,  the  prac- 
tical application  of  the  test  in  many  hundreds  of  thousands 
of  cases  of  syphilis  has  proved  that,  except  in  a  very  few 
easily  recognized  conditions,  the  test  is  specific  for  syphilis 
and  that,  clinically,  it  is  probably  even  more  valuable  than 
it  would  be  if  it  were  a  true  antigen-antibody  reaction,  for 
it  is  doubtful  if  as  large  a  percentage  of  infections  would 
react  positively  to  the  test  if  this  were  true,  judging  from 
the  results  of  complement  fixation  in  other  diseases  which 
are  true  antigen-antibody  reactions. 

Although,  as  generally' performed,  the  Wassermann  test 
is  not  a  true  specific  reaction,  the  work  of  Noguchi^^  and  of 
Nichols  and  the  writer^^  has  proven  that,  with  antigens 
prepared  from  pure  cultures  of  Treponema  pallidum  com- 
plement fixation  can  be  obtained  with  syphilitic  sera,  and 
that  in  such  instances  the  reaction  is  really  a  specific  one, 
due  to  antibodies  in  the  patient's  blood  serum  against  the 
treponema.  This  subject  will  be  discussed  in  a  later  chap- 
ter.    (Chapter  VI.) 


CHAPTER  II 

GENERAL  DESCRIPTION  OF  THE  WASSERMANN 
TEST.  NATURE  OF  THE  REACTION 

As  stated  in  the  preceding  chapter,  Wassermann  and 
his  colleagues  considered  the  complement-fixation  test  for 
syphilis  as  a  true  antigen-antibody  reaction,  the  aqueous 
extract  of  tissue  containing  Treponema  pallidum  furnish- 
ing the  specific  antigen,  while  the  patient's  blood  serum 
contained  the  specific  antibody  or  amboceptor.  This  con- 
ception of  the  reaction  had  to  be  abandoned  when  it  was 
demonstrated  that  aqueous  or  alcoholic  extracts  of  normal 
tissues  could  serve  equally  well  as  an  antigen  in  the  test. 
Therefore,  it  follows  that  the  terms  ''antigen"  and  "ambo- 
ceptor" when  used  in  reference  to  this  test,  except  when 
applied  to  the  hemolytic  system  employed,  are  not  strictly 
accurate,  inasmuch  as  the  antigenic  extracts  are  not  true 
antigens,  their  injection  into  animals  not  being  followed  by 
the  development  of  antibodies ;  while  the  substance  or  sub- 
stances in  the  syphilitic  patient's  serum  which,  in  the  pres- 
ence of  complement  and  the  antigenic  extract,  fix  the  for- 
mer, are  not  true  amboceptors,  for  they  are  not  produced 
by  injection  of  the  antigenic  extracts  employed  in  the  test. 
However,  as  the  reaction  agrees  in  all  essentials  with  really 
specific  complement-fixation  reactions,  and  as  the  antigenic 
extracts  and  the  syphilitic  patient's  serum  act  in  the  same 
manner  as  do  true  antigens  and  antibodies,  it  is  convenient 
to  retain  these  terms  in  speaking  of  the  reaction. 

As  already  stated  the  discovery  of  complement  fixation 
by  Bordet  and  Gengou  was  quickly  utilized  in  the  diagnosis 
of  infectious  diseases  and  the  Wassermann  test  is  simply 
an  adaptation  of  the  principle  to  the  diagnosis  of  syphilis. 
In  performing  the  test  a  hemolytic  system  is  used  as  an 

30 


GENERAL   DESCRIPTION  31 

indicator  and  what  corresponds  with  a  bacteriolytic  system 
is  used  as  the  diagnostic  system.  The  hemolytic  system 
consists  of  a  suspension  of  erythrocytes,  the  antigen;  an 
immune  serum  containing  antibodies  to  the  erythrocytes 
used  as  antigen,  the  amboceptor;  and  fresh  normal  blood 
serum,  the  complement.  The  bacteriolytic  system  of  true 
specific  complement-fixation  reactions  is  replaced  by  the 
syphilitic  system,  as  it  may  be  called,  consisting  of  an  ex- 
tract of  syphilitic  or  normal  tissues,  the  antigen ;  the  syph- 
ilitic patient's  serum,  corresponding  to  the  amboceptor; 
and  complement,  or  fresh  normal  blood  serum. 

These  various  reagents  are  employed  in  exactly  the  same 
manner  as  are  the  hemolytic  and  bacteriolytic  systems  in 
specific  complement-fixation  reactions,  as  will  be  apparent 
from  the  general  description  of  the  test  which  follows : 

It  will  be  remembered  that  in  neither  a  hemolytic  nor  bac- 
teriolytic experiment  will  either  hemolysis  or  bacteriolysis 
be  brought  about  unless  all  three  factors  necessary  for  this^ 
to  occur  are  brought  together ;  i.  e.,  antigen,  amboceptor     .  j 
and  complement  must  be  placed  in  the  same  mixture  before^, 
either  hemolysis  or  bacteriolysis  can  occur.    The  immune 
serum  contains  the  amboceptor  and  this  body  acts  as  an 
intermediary  or  connecting  link  between  the  antigen  and 
complement,  and  when  this  union  occurs  either  hemolysis 
or  bacteriolysis  results,  as  the  case  may  be.    In  theJWas;^^,.  'N  '■■  "^ 
sermann  test  the  patient's  blood  serum,  if  he  is  syphilitic, 
acts  as  the  amboceptor,  the  serum  containing  some  sub- 
stance or  substances  capable  of  uniting  with  antigen  (the 
extract  employed)  and  complement  and  binding  the  latter. 
In  order  to  demonstrate  the  presence  of  this  complement 
binding  substance  in  the  patient's  serum  the  following  pro- 
cedure is  employed,  and  it  is  this  procedure  that  is  known 
as  the  complement-fixation  test  for  syphilis  or  the  Wasser-  tv/ 
mann  test : 

In  a  test  tube  containing  a  suitable  amount  of  normal 
salt  solution  there  is  placed  a  certain  amount  of  the  blood 
serum  from  the  patient  to  be  tested;  a  certain  amount  of 


32 


THE    WASSERMAISTN    TEST 


fresh  normal  blood  serum,  generally  from  a  guinea  pig, 
which  contains  complement ;  and  a  certain  amount  of  an  ex- 
tract of  syphilitic  or  normal  tissues,  the  antigen.  This  mix- 
ture is  allowed  to  stand  in  a  water-bath  at  37°  C.  for  one 
half  hour,  at  the  end  of  which  time  there  are  added  to  the 


Schematic  representation  of  complement  fixation  in  syphilis  or  the  Wassermann  test. 

A.  The  reagents  entering  into  the  reaction;  a,  human  red  blood  corpuscles  or  hemolytic 
antigen;  b,  hemolytic  amboceptor,  or  immune  rabbit  serum;  c,  complement,  or  guinea  pig 
serum;  d,  patient's  blood  serum,  which  may,  or  may  not  contain  syphilitic  antibody; 
e,   syphilitic   antigen   or  antigenic   extract. 

B.  Representing  a  positive  reaction.  The  patient's  blood  serum,  d,  containing  syph- 
ilitic amboceptor,  has  caused  the  union  of  complement,  c,  and  syphilitic  antigen,  e,  so 
that  when  the  hemolytic  antigen,  a,  and  the  hemolytic  amboceptor,  b,  are  added  to  the 
mixture,  no  hemolysis  results,  as  the  complement  has  already  been  fixed  or  absorbed,  as 
shown  in  the  figure. 

C.  Representing  a  negative  reaction.  The  patient's  blood  serum,  d,  containing  no 
syphilitic  amboceptor,  does  not  fix  the  complement  with  the  syphilitic  antigen,  e,  so  that 
when  the  hemolytic  antigen,  a,  and  the  hemolylic  amboceptor,  b,  are  added,  union  occurs 
between  them  and  the   complement,   and  hemolysis   results. 


mixture  a  certain  amount  of  a  suspension  of  human  or 
other  red  blood  corpuscles  and  a  certain  amount  of  the 
blood  serum  of  a  rabbit  imaiunized  to  the  blood  corpuscles 


GENERAL   DESCRIPTIOIsr  33 

employed  in  the  suspension.  The  mixture  is  again  incu- 
bated in  a  water-bath  at  37°  C,  this  time  for  one  hour,  and 
then  placed  in  an  ice  box,  and  the  result  noted  at  the  end  of 
an  hour  or  two. 

If  the  patient  is  suffering  from  syphilis,  the  result  will 
be  that  no  hemolysis  occurs,  and  the  test  is  called  positive, 
while,  if  syphilis  is  not  present,  hemolysis  will  occur,  and 
the  test  is  said  to  be  negative.  The  positive  result  is  due 
to  the  fact  that  during  the  first  incubation  the  substance  in 
the  patient's  blood  serum,  corresponding  to  a  specific  am- 
boceptor, unites  the  antigen,  or  extract  used  in  the  test 
and  the  complement,  thus  binding  the  latter,  so  that  when 
later  the  erythrocytes  and  immune  serum  or  amboceptor 
against  them  is  added,  there  is  no  complement  left  to  unite 
with  them  and  therefore,  hemolysis  can  not  occur.  In 
case  that  the  result  is  negative,  this  is  due  to  the  fact  that 
the  patient's  blood  serum  contained  no  substance  capable 
of  uniting  the  antigenic  extract  and  complement,  so  that 
when  later  the  erythrocytes  and  the  immune  serum  to  them 
were  added,  the  amboceptor  in  the  immune  serum  caused 
the  union  of  the  erythrocytes  and  the  complement  and 
hemolysis  resulted,  the  complement  having  been  left  free 
in  the  mixture,  owing  to  nonunion  during  the  first  incuba- 
tion. 

From  this  brief  description  of  the  Wassermann  test  it 
will  be  noted  that  it  corresponds  in  every  way  as  regards 
technic  and  results  with  specific  complement-fixation  tests, 
but  that  instead  of  a  specific  bacteriolytic  antigen  there  is 
substituted  a  nonspecific  tissue  extract  which  serves  as  an 
antigen  and  which  acts  in  the  same  manner  as  does  the 
specific  antigen  employed  in  bacteriolytic  experiments, 
while  the  complement-fixing  substance  or  substances  in  the 
syphilitic  patient's  blood  serum  act  in  the  same  manner  as 
does  the  amboceptor  in  the  immune  serum  in  bacteriolytic 
experiments.  When,  however,  an  extract  of  a  pure  cul- 
ture of  Treponema  pallidum  is  used  as  an  antigen  there  is 
no  difference  between  the  Wassermann  test  and  other  com- 


34  THE   WASSERMAIsrisr   TEST 

plement  fixation  reactions,  as  in  this  case  the  complement 
fixation  is  the  result  of  a  true  antigen-antibody  reaction. 
Unfortunatel}'',  the  antigens  prepared  from  pure  cultures 
of  Treponema  'pallidum  are  very  unreliable  in  practice,  so 
that  they  can  not  be  used  for  this  purpose,  and  the  nonspe- 
cific antigens  have  to  be  depended  upon  in  the  test. 

Nature  of  the  Wassermann  Reaction 

The  exact  nature  of  the  Wassermann  reaction  is  still  a 
mystery,  despite  an  immense  amount  of  work  that  has  been 
done  in  the  endeavor  to  solve  the  problem.  The  fact  that  ex- 
tracts of  tissues  which  do  not  contain  Treponema  pallidum 
serve  equally  well  as  antigens  as  those  that  do,  and  even 
better  than  antigens  prepared  from  pure  cultures  of  the 
organism,  proves  that  the  reaction  is  not  a  true  antigen- 
antibody  reaction.  The  experiments  of  Noguchi^^  demon- 
strating that  the  active  antigenic  principles  in  the  normal 
tissue  extracts  were  lipoids  and  his  conclusion  that  the  re- 
action depends  upon  lipotropic  substances  in  the  patient's 
blood  serum,  showed  that  the  cause  of  the  reaction  is 
closely  connected  with  the  chemistry  of  the  lipoids,  and 
this  is  about  all  that  we  can  claim  to  know  regarding  its 
exact  nature  to  date,  although  many  ingenious  and  inter- 
esting theories  have  been  advanced  in  explanation  of  the 
phenomenon. 

Citron^*  adopted  the  hypothesis  that  the  antibody  pro- 
ducing antigen  is  a  toxolipoid  and  has  proposed  the  name 
*'Luesargine"  for  the  syphilitic  antibodies.  In  conJTinc- 
tion  with  Miinck  he  claims  to  have  demonstrated  that  the 
true  syphilitic  antigen  is  contained  only  in  aqueous  ex- 
tracts of  syphilitic  tissues  and  that  only  rabbits  inoculated 
with  such  extracts  produce  antibodies  that  react  positively 
with  alcoholic  extracts  of  normal  organs,  but  his  work  has 
not  been  confirmed. 

Schmidt^^  considers  that  the  reaction  is  colloidal  in  na- 
ture, depending  upon  the  union  of  the  colloids  in  the  anti- 


NATURE   OF   THE   REACTION  35 

genie  extracts  and  the  serum  globulins  in  the  patient's 
blood  serum,  which  are  increased  in  amount  and  changed 
in  nature  in  syphilitic  patients. 

Levaditi  and  Yamanouchi"^  believe  that  the  reaction  de- 
pends upon  the  union  of  two  colloidal  factors,  one  present 
in  the  patient's  blood  serum  and  nonproteid  in  character, 
the  other  consisting  of  lipoidal  substances  in  the  extracts 
used  as  antigen. 

Weil  and  Braun^''  conclude  that  during  the  progress  of 
the  disease  certain  tissue  products,  lipoidal  in  nature,  are 
absorbed,  and  give  rise  to  antibodies  which  will  react  in 
vitro  with  the  lipoids  present  in  the  antigenic  extracts,  the 
so-called  autoantibody  theory.  This  theory  is  somewhat 
similar  to  Citron's,  both  resting  upon  the  belief  that  in  the 
tissues  of  the  patient  there  are  produced  antibodies  to  the 
lipoids  in  the  antigenic  extracts,  these  antibodies  being  pro- 
duced by  the  absorption  in  the  body  of  albumin-lip oid  sub- 
stances produced  by  the  growth  of  Treponema  pallidum, 
and  the  subsequent  production  of  antibodies  against  these 
substances. 

BergeP^  considers  that  the  Wassermann  reaction  may  be 
due  to  the  action  of  a  specific  lipase  which  is  produced  iu 
the  syphilitic  patient  against  "lues-lipoids."  This  theory 
can  not  hold  in  the  light  of  the  facts  demonstrating  that 
the  reaction  occurs  with  extracts  prepared  from  normal  or- 
gans, when  used  as  antigen. 

Manwaring^^  thinks  that  in  guinea  pig  blood  serum  there 
is  a  proteolytic  ferment  which  may  weaken  or  destroy  com- 
plement and  that  the  fixation  reaction  is  due  to  substances 
present  both  in  the  antigenic  extracts  and  syphilitic  pa- 
tient's serum  which  increase  this  effect.  Bruck  and  Stern^'' 
believe  that  the  reaction  is  due  to  the  interaction  of  the 
lipoid  bodies  in  the  antigenic  extracts  and  similar  bodies 
in  the  patient's  blood  serum;  while  Peritz^^  concludes  that 
it  is  due  to  some  substance  in  the  blood  serum  that  has  an 
affinity  for  lecithin,  which  is  always  contained  in  the 
extracts. 


36  THE   WASSERMANN   TEST 

None  of  the  various  theories  advanced  to  account  for 
this  reaction  have  been  accepted  as  the  true  explanation  of 
the  process  of  complement  fixation  in  syphilis  and  none  of 
them  rest  upon  sufficient  experimental  evidence  to  be  ac- 
cepted as  the  explanation.  The  most  that  can  be  said  re- 
garding the  reaction  is  that  it  is  due  to  an  interaction  be- 
tween lipotropic  bodies  present  in  the  blood  serum  of  syph - 
ilitics  and  lipoids  in  the  antigenic  extracts.  A  mixture  of 
a  lipoid  extract  and  syphilitic  blood  will  absorb  a  large 
amount  of  complement,  so  that  it  follows  that  the  substance 
or  substances  in  the  serum  must  be  lipotropic  in  nature. 
This  lipotropic  substance  is  more  constantly  present  in  the 
blood  of  syphilitics  than  in  other  diseases,  and  is  prac- 
tically specific  for  that  disease.  Kesearches  have  conclu- 
sively proved  that  the  lipotropic  substance  or  substances 
are  due  to  unknown  changes  in  the  tissues  or  blood  serum 
brought  about  by  the  actual  presence  in  the  tissues  of  liv- 
ing Treponema  pallidum,  and  that  the  reaction  disappears 
with  the  cure  of  the  infection,  so  that  its  presence  is  proof 
of  the  existence  of  syphilis  even  though  months  or  years 
may  have  passed  without  definite  clinical  symptoms  having 
been  observed. 

Although  the  Wassermann  reaction,  as  the  test  is  usually 
performed,  is  not  a  true  antigen-antibody  reaction,  there 
is  proof  that  in  the  blood  of  syphilitics  true  antibodies 
against  Treponema  pallidum  are  formed  which  will  fix 
complement  in  the  presence  of  antigens  prepared  from 
pure  cultures  of  the  organism. 

The  first  experiments  along  this  line  were  initiated  by 
Schereschewsky,^"  who  obtained  positive  reactions  in  syph- 
ilitics by  using  antigens  made  from  an  impure  culture  of 
what  he  regarded  as  Treponema  pallidum.  Owing  to  the 
doubt  attending  the  impurity  of  the  culture  and  as  to  the 
exact  species  of  treponema  with  which  he  worked,  Scheres- 
chewsky's  results  were  not  conclusive,  and  it  was  not  until 
Noguchi^^  cultivated  in  pure  culture  the  organism  of  syph- 
ilis and  employed  antigens  prepared  from  these  pure  cul- 


NATURE    OF   THE   REACTION  37 

tures,  that  it  was  proved  that  true  complement  fixation  did 
occur  in  syphilis.  Noguchi^*  demonstrated,  that  by  using 
aqueous  extracts  of  pure  cultures  of  Treponema  pallidum 
as  antigen,  it  was  possible  to  secure  complement  fixation  in 
certain  cases  of  the  disease,  but  that  the  reaction  did  not 
occur  as  consistently  as  when  nonspecific  antigens  were 
employed  nor  did  it  occur  at  all  stages  of  the  disease. 
Noguchi  immimized  rabbits  with  the  pure  cultures,  using 
the  aqueous  pallidum  extracts  for  injection,  and  found  that 
their  blood  bound  complement  when  the  pallidum  extract 
was  used  as  antigen  but  that  negative  results  were  ob- 
tained when  extracts  of  normal  tissues  were  used  as  anti- 
gens. These  results  proved  that  the  blood  serum  of  the 
immunized  rabbits  contained  a  real  antibody  against  the 
syphilitic  toxin  contained  in  the  pallidum  extract  used  in 
immunization.  He  found,  however,  that  Avhile  the  blood 
serum  of  rabbits  with  an  active  syphilitic  orchitis,  gave 
positive  complement  fixation  with  the  extracts  of  normal 
tissue,  rich  in  lipoids,  it  did  not  bind  complement  when  the 
pallidum  extract  was  used.  He  explained  this  result  as 
follows  :^^ 

The  findings  show  that  the  immune  serums  contain  the  specific  antibodies 
for  the  pallidum  but  not  for  the  lipoids,  and  that  in  the  serums  of  syphilitic 
rabbits  (active  stage)  there  is  an  abundance  of  the  lipotropic  substance 
(probably  an  enzyme)  capable  of  rendering  the  complement  inactive  (so- 
called  fixation)  in  the  presence  of  certain  lipoids,  but  too  little  specific  anti- 
bodies to  bind  complement  vrith  the  pallidum  antigen. 

When  pure  pallidum  antigens  were  used  with  human 
syphilitic  blood  serum,  Noguchi  obtained  similar  results, 
the  early  stages  of  the  disease,  primary  and  secondary, 
seldom  reacting  with  the  specific  antigen,  while  in  the  ter- 
tiary, latent,  and  late  congenital  cases  the  blood  serum  fre- 
quently gave  a  positive  result.  Because  of  these  diverse 
results  in  the  various  stages  of  the  disease,  when  the  pure 
pallidum  antigen  Avas  used,  Noguchi  concluded  that  a  posi- 
tive result  with  the  pallidum  antigen  indicated  the  forma- 
tion of  true  antibodies  in  the  blood  of  the  patient,  and  was, 


do  THE   WASSERMAI^N   TEST    . 

therefore,  of  favorable  omen,  while  a  negative  reaction  in- 
dicated their  absence. 

In  1912,  the  writer  and  Nichols,^®  using  antigens  made  by 
extracting  pure  cultures  of  Treponema  pallidum  with  alco- 
hol, obtained  complement  fixation  in  all  stages  of  syphilis, 
but  positive  reactions  were  also  obtained  with  syphilitic 
blood  serum  "uith  antigens  made  from  pure  cultures  of 
Treponema  pertenue,  Treponema  microdentium,  and  Spi- 
roclieta  refringens,  while  antigens  made  from  pure  cul- 
tures of  organisms  like  Bacillus  typhosus  and  Spirillum 
cliolercB  gave  negative  results.  We  concluded  that  the  pos- 
itive reactions  ^dth  the  antigens  prepared  from  the  organ- 
isms mentioned  was  in  the  nature  of  a  group  reaction,  and 
that  the  practical  value  of  the  test  Avhen  the  pallidum  anti- 
gen was  used  was  small,  OAAdng  to  the  fact  that  many  cases 
of  the  disease  gave  a  negative  reaction,  even  during  the 
late  stages  of  the  infection.  Our  results  have  since  been 
confirmed  by  Kolmer,  AVilliams  and  Laubaugh"  and  other 
investigators. 

A^Tiile  it  may  be  considered  as  proved  that  antigens  pre- 
pared from  pure  cultures  of  Treponema  pallidum  are  unre- 
liable and  of  little  value  in  the  practical  application  of  the 
Wassermann  test,  their  use  has  demonstrated  that  a  spe- 
cific fixation  of  complement  may  be  obtained  in  syphilis 
when  such  antigens  are  used  in  the  complement-fixation 
test.  This  fact  renders  it  very  probable  that  antigens  pre- 
pared from  foetal  syphilitic  liver,  rich  in  treponemas,  may 
give  positive  results  in  rare  instances  where  extracts  of 
normal  tissues  will  fail,  and  the  use  of  the  two  kinds  of 
antigen  in  practice  has  sho^^m  that  this  is  the  case.  The 
writer  is,  therefore,  of  the  opinion  that  the  A¥assermann 
reaction,  when  an  extract  of  foetal  syphilitic  liver  is  used 
as  antigen,  is  of  a  dual  nature,  in  some  instances,  consist- 
ing of  a  true  antigen-antibody  reaction  between  specific 
antibodies  in  the  patient's  blood  serum  and  specific  sub- 
stances in  the  antigen;  and  a  nonspecific  reaction  between 
the  lipotropic  substance  in  the  patient's  serum  and  lipoids 


NATURE   OF   THE   REACTIOlSr  39 

in  the  antigen.  When  an  extract  of  normal  tissues  is  used 
as  antigen  the  reaction  is  due  to  the  lipoids  in  the  antigen 
and  lipotropic  substances  in  the  patient's  blood  serum,  and, 
in  such  instances,  the  possible  reaction  between  true  anti- 
bodies and  the  specific  substance  in  the  antigen  is  lost,  and 
the  reaction  may  be  negative  instead  of  positive.  "While, 
in  practice,  in  the  writer's  experience,  this  result  very 
rarely  occurs,  it  is  considered  best,  if  possible,  to  make  use 
of  both  varieties  of  antigen  when  performing  the  Wasser- 
mann  test. 


CHAPTEE  III 

FACTORS  WHICH  INFLUENCE  THE  RESULT 
OF  THE  TEST 

The  Wassermann  test  is  quantitative  in  nature  and  un- 
less all  of  the  reagents  are  carefully  titrated  at  frequent 
intervals  and  accurately  used,  erroneous  results  are  sure 
to  occur.  The  quantity  of  blood  serum  to  be  tested;  its 
proper  inactivation ;  the  dose  of  complement  and  its 
source;  and  the  amount  of  amboceptor  and  antigen  to  be 
used;  are  aU  important  factors  in  the  test  and  variations 
from  the  proper  amount  of  each  will  be  followed  by  faulty 
results,  as  will  be  shown  in  the  chapter  devoted  to  technic. 

However,  aside  from  the  reagents  employed  in  the  test 
there  are  certain  factors  which  have  a  very  marked  influ- 
ence upon  its  result  and  which  should  be  understood  by 
physicians  using  this  valuable  diagnostic  measure.  These 
factors  are:  The  influence  of  the  ingestion  of  alcohol;  the 
influence  of  certain  bacteria  in  the  blood  serum ;  and  varia- 
tions in  the  amount  of  complement  inhibiting  substance  in 
the  blood  serum  to  be  tested. 

The  Influence  of  the  Ingestion  of  Alcohol. — In  1911, 
Major  Nichols,  of  the  Medical  Corps  of  the  Army,  and 
the  writer'^^  called  attention  to  the  effect  that  the  inges- 
tion of  considerable  amounts  of  alcohol  has  upon  the  re- 
sults of  the  Wassermann  test.  It  was  found  that  the  in- 
gestion of  alcohol  in  the  form  of  beer  or  whiskey,  and  in 
considerable  amounts,  might  convert  a  strongly  positive 
serum  into  a  negative  one,  if  the  test  were  made  within 
twenty-four  hours  after  the  ingestion  of  the  alcohol,  and 
that  the  blood  might  remain  negative  for  as  long  as  three 
days,  although  it  generally  became  positive  again  mthin 
twenty-four  hours.    This  fact  was  discovered  by  the  writer 

40 


FACTORS   INFLUENCIN'G   RESULT   OF   TEST  41 

while  making  routine  examinations  of  the  blood  of  a  cer- 
tain individual  who  was  being  treated  for  syphilis  by 
Major  Nichols,  and  who  had  presented  several  strong 
positive  reactions  before  the  first  negative  one  which  was 
due  to  the  ingestion  of  alcohol,  and  further  research  by 
Nichols  and  myself  proved  that  this  drug  has  a  very  po- 
tent influence  in  weakening  or  destroying  the  lipotropic 
substances  in  the  patient's  blood  serum  which  apparently 
cause  the  Wassermann  reaction. 

The  case  in  which  this  phenomenon  was  first  observed  is 
of  special  interest  in  this  connection  and  the  clinical  his- 
tory is  here  given : 

Male,  41  years  of  age.  Infected  with  syphilis  in  1904.  Now  has  palmar 
lesions.  February  25,  1911,  0.5  gm.  of  salvarsan  was  administered  intra- 
venously, the  Wassermann  test  before  administration  giving  a  plus  reaction. 
Between  this  date  and  April  7th,  1911,  his  blood  was  tested  five  times,  the 
reaction  four  times  being  double-plus,  and  the  last  test,  on  April  7th,  being 
plus.  (Double-plus  here  equals  four-plus  of  most  authorities.)  April  8th 
his  blood  was  again  tested  and  was  found  to  be  negative.  The  result  was 
unusual  and  on  inquiry  it  was  learned  that  on  the  night  of  April  7th  he 
drank  about  700  c.c.  of  Munich  beer.  His  blood  was  tested  again  on  April 
12th,  and  gave  a  double-plus  (four-plus  of  some  authors)  reaction.  Be- 
lieving that  the  beer  might  have  had  something  to  do  with  the  sudden 
disappearance  of  the  positive  reaction  in  this  case,  we  requested  that  he 
repeat  the  beer  on  the  night  of  April  14th,  which  he  did,  and  on  the  morn- 
ing of  April  15th  the  reaction  was  again  negative.  On  the  17th  of  April  it 
had  become  plus  and  on  the  22nd  again  was  double-plus. 

As  it  was  evident  that  the  negative  result  of  the  Wasser- 
mann test  was  due,  in  this  case,  to  the  ingestion  of  the  beer, 
we  decided  to  repeat  the  experiments  on  other  cases.  We 
selected  nine  syphilitic  patients,  who  before  the  adminis- 
tration of  alcohol  gave  a  double-plus  reaction,  and  Table  I 
gives  the  results  obtained  in  these  patients.  It  will  be  re- 
membered that  in  this  table,  the  double-plus  reaction  means 
absolute  inhibition  of  hemolysis,  and  is  equivalent  to  the 
four-plus  reaction  of  most  writers. 

Of  the  9  cases  included  in  this  table,  3  were  in  the  sec- 
ondary stage  of  the  disease  and  6  were  in  the  tertiary 
stage.    The  quantity  of  alcohol  administered  varies  some- 


42 


THE   WASSERMAISTN    TEST 


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FACTORS  INFLUENCING  RESULT  OF  TEST         43 

what  but  in  every  case  the  positive  reaction  disappeared 
after  its  administration.  In  most  of  the  cases  the  blood 
was  tested  one  hour  before  the  administration  of  the  al- 
cohol was  begun  as  well  as  afterward.  A  study  of  the  ta- 
ble shows  that  in  5  of  the  cases  the  reaction  was  found  to 
be  negative  one  hour  after  the  last  dose  of  the  alcoholic 
liquor  had  been  administered,  and  that  in  all  the  cases  the 
reaction  remained  negative  for  several  hours.  In  cases  4, 
5,  and  6  the  reaction  remained  negative  for  over  24  hours 
while  in  case  9  it  remained  negative  for  three  days  and  did 
not  become  double-plus  again  until  over  four  days  had 
elapsed  from  the  time  the  alcohol  was  administered.  Two 
of  the  patients  had  received  salvarsan  before  the  admin- 
istration of  the  alcohol,  but  in  one  the  drug  had  been  given 
so  long  before  that  it  could  not  have  had  any  effect  upon 
the  reaction,  while  in  the  other  the  fact  that  the  reaction 
became  double-plus  again  after  becoming  negative  from 
the  alcohol,  proves  that  the  salvarsan  had  nothing  to  do 
with  its  disappearance. 

That  the  disappearance  of  the  reaction  after  the  inges- 
tion of  alcohol  is  not  due  to  the  alcohol  itself,  which  may 
be  present  in  the  blood  serum,  is  proved  by  the  fact  that 
a  much  larger  quantity  of  alcohol  is  necessary  to  produce 
hemolysis,  as  proved  by  experiment,  than  could  possibly 
be  present  in  the  blood  serum  of  the  patients  tested.  Thus, 
we  found  that  it  requires  at  least  0.15  c.c.  of  absolute  alco- 
hol to  produce  complete  hemolysis  of  1  c.c.  of  a  1  per  cent 
suspension  of  human  red  blood  corpuscles  within  the  time 
used  in  the  Wassermann  test  for  incubation  of  the  hem- 
olytic system,  whereas  only  0.08  c.c.  of  the  patient's  blood 
serum  was  used  in  each  test.  It  is  thus  evident  that  if  the 
amount  of  blood  serum  used  had  been  actually  absolute  al- 
cohol the  quantity  would  have  been  insufficient  to  have  pro- 
duced hemolysis. 

From  these  experiments,  which  have  been  confirmed 
by  many  investigators  since  our  original  publication, 
it    is    evident    that    alcohol    may    render  inert  the  sub- 


44  THE   WASSEKMANlsr   TEST 

stance  or  substances  in  the  blood  serum  of  syphilitics 
which  react  with  lipoids  in  the  antigenic  extracts,  and  thus 
a  strongly  positive  serum  may  give  a  negative  result.  For 
this  to  occur,  the  alcohol  must  be  taken  in  considerable 
quantity  and  probably  within  twenty-four  hours,  or  at 
most  three  days  before  the  test  is  made,  but  it  should 
be  remembered  that  smaller  amounts  of  alcohol  may  ren- 
der weak  reactions  negative,  so  that  cases  which  should 
present  a  single  plus  reaction  will  often  react  nega- 
tively after  even  moderate  amounts  of  alcoholic  liquors 
have  been  ingested.  It  is  thus  evident  that  very  careful 
inquiry  should  be  made  regarding  the  use  of  alcoholic  liq- 
uors before  taking  blood  for  a  Wassermann  test  from  any 
patient,  and  if  there  is  a  history  of  indulgence  in  such  liq- 
uors within  from  twenty-four  to  thirty-six  hours  previ- 
ously, the  taking  of  the  specimen  should  be  postponed  and 
alcoholics  forbidden  until  at  least  two  days  have  passed. 
The  neglect  of  this  precaution  has  often  resulted  in  errone- 
ous results  and  cases  that  should  have  been  strongly  posi- 
tive have  been  reported  as  negative  or  doubtful.  When 
the  test  is  used  as  a  control  of  treatment  alcohol,  even  in 
small  amounts,  should  be  forbidden  for  some  days  before 
the  blood  is  collected,  or  the  weak  reactions  often  observed 
in  treated  cases  will  be  entirely  missed,  thus  obscuring  the 
real  condition  of  the  patient  as  regards  his  serological 
status. 

From  what  has  been  said,  it  follows  that  a  negative  Was- 
sermann reaction,  after  the  ingestion  of  alcoholic  liquors, 
possesses  no  value  whatever,  and  an  immense  amount  of 
harm  has  doubtless  been  done  by  lack  of  care  in  ascertain- 
ing whether  patients  have  indulged  in  such  liquors  within 
a  short  time  before  the  blood  was  collected.  Our  observa- 
tions, as  well  as  those  of  others,  both  in  this  country  and 
abroad,  have  proved  that  the  effect  of  alcohol  upon  the 
Wassermann  reaction  is  very  important  from  a  practical 
standpoint,  and  one  which  should  not  be  forgotten  when 


TACTORS   INFLUESrCING   RESULT   OE   TEST  45 

patients  come  to  the  physician  for  the  collection  of  blood 
for  the  test. 

The  Influence  of  Certain  Bacteria,  when  Developing  in 
the  Blood  Serum,  upon  the  Results  of  the  Test. — It  is  a  well- 
known  fact  that,  upon  standing  for  varying  lengths  of  time, 
some  human  blood  sera  develop  anticomplementary  sub- 
stances, which  cause  inhibition  of  hemolysis  in  both  the 
tubes  containing  antigen  and  the  control  tubes  used  in  the 
Wassermann  test.  Most  of  these  bodies  are  destroyed  by 
heating  the  blood  serum  for  one-half  hour  at  56°  C,  but, 
in  some  instances,  they  still  persist  after  heating,  and  in 
such  sera  one  is  unable  to  read  the  result  of  the  test  owing 
to  inhibition  occurring  in  both  the  antigen  and  control 
tubes.  From  these  observations  it  is  evident  that  these 
anticomplementary  bodies  are  of  two  kinds,  thermolabile 
and  thermostable,  and  that  the  inactivation  of  the  blood 
serum  by  heating  enables  us  to  destroy  only  the  thermo- 
labile group. 

While  the  exact  nature  of  these  anticomplementary 
bodies  is  still  doubtful,  the  writerias  shown^^  that  some 
of  them  are  produced  by  the  growth  of  bacteria  in  the  blood 
serum  to  be  tested,  if  the  serum  is  kept  for  some' time.  Not 
only  will  certain  bacteria  produce  anticomplementary 
bodies  that  cause  inhibition  of  hemolysis  in  both  the  anti- 
gen and  control  tubes  of  the  test,  but  certain  species  also 
possess  the  power  of  causing  inhibition  of  hemolysis  in  the 
antigen  tube  alone,  thus  giving  rise  to  a  nonspecific  reac- 
tion in  the  sera  containing  them.  It  is  these  species  that 
are  of  special  importance  in  the  application  of  the  Wasser- 
mann test  and  which  mil  here  be  discussed. 

While  carrying  on  some  comparative  tests  of  blood  se- 
rum with  another  laboratory  it  was  noted  that  in  one  in- 
stance which  gave  a  positive  reaction  in  the  writer's  lab- 
oratory and  a  negative  one  at  another  laboratory  situated 
five  days'  journey  distant,  the  blood  serum  when  received 
was  cloudy  in  appearance  and  had  a  bad  odor.  As  it  was 
known  to  be  infected  and  as  it  was  thought  possible  that 


46 


THE   WASSERMANN   TEST 


bodies  developed  in  the  serum  by  the  growth  of  the  bac- 
teria might  be  the  cause  of  the  difference  in  results,  this 
serum  was  plated  out  upon  agar-agar  and  cultures  of  the 
following  organisms  were  isolated :  Staphylococcus  aureus; 
a  large  Staphylococcus  albus;  a  small  Staphylococcus  albus, 
a  large  spore-bearing  bacillus  of  the  subtilis  tj^e;  and  a 
very  minute  diplobacillus. 

In  order  to  determine  whether  any  of  these  bacteria 
were  responsible  for  the  reaction  obtained  with  this  blood 
serum  the  following  experiments  were  undertaken  to- 
gether mth  experiments  with  a  stock  culture  of  Staphy- 
lococcus aureus,  Staphylococcus  citreus,  and  Streptococcus 
pyogenes.  The  same  technic  was  followed  in  these  exper- 
iments as  was  used  in  performing  the  Wassermann  test, 
the  mixtures  of  serum  and  bacteria  being  substituted  for 
the  patient's  blood  serum. 

Experiment  1. — An  emulsion  in  normal  salt  solution  was  made  from  a 
pure  culture  of  each  organism  isolated  from  the  blood  serum,  and  0.08  c.c.  of 
this  emulsion  was  placed  in  1  c.c.  of  a  1  per  cent  suspension  of  human  red 
blood  corpuscles,  together  with  0.08  c.c.  of  an  inactivated  normal  serum. 
Table  II  gives  the  result  of  this  experiment. 


Table  II 
Results  of  Complement-'Flxatiok  Tests  in  Experiment  No.  1 


Results 

Controls 

NAME  OF  BACTERIUM 

ANTIGEN 
TUBE 

CONTROL 
TUBE 

BACTERIA 
ALONE 

SERUM 
ALONE 

ORIGIN  OF 
BACTERIUM 

S.  albus,  large 
S.  alius,  small 
S.  aureus. 
Bacillus  (Spore) 
Diplobacillus 
Combined  bacteria 

- 

- 

_ 

- 

Isolated 
from 
serum  in 
all  cases 

From  these  results  it  is  evident  that  merely  adding  an 
emulsion  of  the  cultures  of  these  bacteria  to  normal  blood 
serum  is  without  effect  upon  the  result  of  the  complement- 
fixation  test,  as  all  of  the  mixtures  gave  a  negative  reaction. 

Experiment  2. — This  experiment  was  identical  with  Experiment  1,  except 
that  the  mixtures  of  serum  and  bacteria  were  inactivated  at  56°  C.  for  one-half 


EACTOKS   INFLUENCIlSrG    RESULT    OF   TEST 


47 


hour  before  being  tested.  The  mixtures  of  bacteria  and  serum  in  this  experi- 
ment all  gave  a  negative  reaction  with  the  exception  of  the  mixture  of  blood 
serum  and  the  diplobacillus,  which  resulted  in  a  plus  reaction,  the  heating 
resulting  in  the  liberation  of  enough  inhibitory  substances  to  cause  this  type 
of  reaction  in  both  the  antigen  and  control  tubes.  Of  course  this  reaction 
would  have  caused  no  confusion  so  far  as  the  diagnosis  of  syphilis  is  concerned, 
as  the  fact  that  the  inhibition  occurred  in  the  control  tube  as  well  as  in  the 
antigen  tube  would  have  thrown  the  test  out  entirely  and  another  specimen 
would  have  been  requested. 

Experiment  3. — Specimens  of  normal  serum  were  inoculated  with  each  of 
the  bacteria  isolated  from  the  serum  in  question  as  well  as  with  the  stock  cul- 
tures and  allowed  to  stand  at  room  temperature  for  one  week  before  being 
tested.     Table  III  gives  the  results  of  this  experiment: 

Table  III 

Eesults  of  Complement-Fixation  Tests  in  Experiment  No.  3 


NAME  OF 
BACTERIUM 

Result 

CONTROL, 
SERUM 
ALONE 

ANTIGEN 
TUBE 

CONTROL 
TUBE 

ORIGIN  OF 
BACTERIUM 

S.  albus,  large 
8.  albus,  small 
S.  aureus. 
Bacillus  (spore) 
Diplobacillus 
S.  OAireus 
S.  dtreus 
S.  pyogenes 

-1- 
+  + 
+  -1- 

+  + 

1      1     1     1     i     1     1     1 

Isolated  from  serum 
11         <  I         i  ( 

i  i         it         1  ( 

It           It           t  e 

(  t              c  t              t  ( 

Stock  culture 
1 1         It 

t  e            it 

It  is  evident,  by  consulting  the  table,  that  after  incuba- 
tion in  human  blood  serum  for  a  week  at  room  temperature 
the  large  S.  albus  isolated  from  the  blood  serum  under 
discussion  gave  a  plus-minus  reaction  in  the  antigen  tube, 
while  the  diplobacillus  isolated  from  the  same  serum  gave 
a  plus  reaction  in  the  antigen  tube  and  a  double-plus  re- 
action in  the  control  tube.  The  stock  8.  aureus  gave  a 
double-plus  reaction  in  the  antigen  tube,  while  the  stock 
culture  of  8.  citreus  caused  total  inhibition  of  hemolysis  in 
both  antigen  and  control  tubes. 

From  this  experuuent  it  is  evident  that  the  stock  8. 
aureus,  growing  in  normal  blood  serum  at  room  tempera- 
ture for  one  week  produced  some  substance  or  substances 
in  the  serum  which  gave  a  false  positive  reaction. 

Experiment  4. — This  experiment  was  identical  with  Experiment  3,  except 
that  the  mixtures  of  bacteria  and  serum  were  incubated  at  37°  C.  for  24  hours 


48 


THE   WASSERMANN   TEST 


and  then  allowed  to  stand  at  room  temperature  for  one  week,  before  being 
tested.     Table  IV  gives  the  results  of  this  experiment: 

Table;  IV 

EESULTS    OF    CoMPLEMENT-riXATION    TESTS    IN    EXPERIMENT    NO.    4 


Besult 

CONTROL 
SERUM 
ALONE 

ORIGIN  OF 
BACTERIUM 

NAME  OF 
BACTERIUM 

ANTIGEN 
TUBE 

CONTROL 

TUBE 

S.  alhus,  large 

+  + 

_ 

- 

Isolated  from  serum 

S.  albus,  small 
S.  aureus 

+  + 

- 

: 

a            a            11 

Bacillus  (spore) 

_ 

- 

- 

Diplobacillus 
S.  aureus 

+  + 
+  + 

+  + 

— 

Stock  culture 

S.  citreus 
S.  pyogenes 

+  + 
+  + 

+  + 

- 

a           It 

From  this  experimental  record  it  will  be  noted  that  no 
less  than  four  of  the  organisms  tested  gave  a  double-plus 
(four-plus)  positive  reaction  in  the  antigen  tube  alone, 
while  two  inhibited  hemolysis  completely  in  both  the  anti- 
gen and  control  tubes.  It  would  appear  that  the  24  hours 
at  incubator  temperature  lead  to  an  increase  in  the  inten- 
sity of  the  reaction,  due  to  a  richer  development  of  the 
bacteria  in  the  sera. 

Considering  the  experiments  as  a  whole  it  is  perfectly 
evident  that  a  positive  complement-fixation  reaction  may 
be  obtained  in  a  normal  serum  if  certain  species  of  bacteria 
are  enabled  to  develop  within  it.  That  every  strain  of  a 
bacterial  species  will  not  produce  this  result  is  shown  by 
the  fact  that  while  a  double-plus  reaction  was  obtained  in 
the  serum  infected  with  the  stock  S.  aureus  and  with  the 
S.  aureus  isolated  from  the  serum  under  discussion,  a  nega- 
tive result  was  later  obtained  Avith  an  S.  aureus  isolated 
from  a  blood  serum  that  gave  a  negative  reaction. 

It  is  also  evident  that  temperature  has  much  to  do  mth 
the  ability  of  the  bacteria  to  produce  the  positive  reaction, 
for  in  the  mixtures  of  serum  and  bacteria  kept  for  24 
hours  at  incubator  temperature,  the  reactions  were  most 
marked,  while  two  of  the  organisms  which  did  not  give 
a  reaction  when  growing  in  the  blood  serum  at  room  tem- 


FACTORS   INFLUEITCING   EESULT   OF    TEST  49 

perature,  gave  a  strongly  positive  reaction  Avhen  incnbated. 
Inactivation  also  appears  to  favor  the  production  of  the 
false  positive  reaction  in  sera  which  are  contaminated  with 
certain  bacteria. 

As  regards  the  serum  under  discussion,  it  is  evident  that 
the  positive  result  could  have  been  caused  by  either  the 
large  S.  alhus  or  the  S.  aureus,  or  both,  according  as  the 
conditions  at  the  time  of  examination  were  favorable  to  the 
production  of  inhibitory  substances  by  one  or  both  of  these 
organisms. 

The  fact  that,  under  certain  conditions,  such  common 
bacteria  as  staphylococci  and  streptococci,  Avhen  growing 
in  normal  blood  seram,  may  give  rise  to  a  positive  Was- 
sermann  reaction,  is  of  great  practical  importance,  and 
one  might  conclude  from  these  experiments  that  only  fresh 
blood  serum  should  be  used  in  making  complement-fixation 
tests.     However,  if  proper  precautions  are  taken  in  the 
collection  of  specimens,  there  is  no  danger  of  bacterial 
contamination,  and  it  is  our  experience  that,  unless  such 
contamination  occurs,  a  positive  result  will  not  be  obtained 
in  normal  serum,  even  when  kept  for  a  considerable  period 
of  time.     Personal  experiments  have  demonstrated  that 
blood  sera  may  be  kept  at  room  temperature  for  as  long 
as  one  month  without  danger  of  a  negative  serum  becom- 
ing positive,  provided  there  is  no  bacterial  contamination 
in  the  serum.    While,  in  such  specimens,  anticomplemen- 
tary bodies  frecjuently  develop,  after  they  have  been  kept 
for  a  week  or  more,  these  bodies  cause  inhibition  of  hem- 
olysis in  both  the  antigen  and  control  tubes  of  the  serum, 
and  thus  while  destroying  the  value  of  the  test,  this  type 
of  reaction  does  not  cause  any  confusion  in  the  diagnosis 
of  the  disease.    However,  these  observations  emphasize  the 
importance  of  collecting  specimens  for  the  Wassermann 
test  under  aseptic  precautions  if  the  sera  can  not  be  ex- 
amined within  24  hours  after  collection. 


50  THE   WASSERMANN"   TEST 

The  Influence  of  Variations  in  the  Strength  of  the  Re- 
action in  Untreated  Cases  of  Syphilis  upon  the  Results  of 
the  Test. — During  the  routine  examinations  of  blood  sera 
for  the  Wassermann  test,  the  writer  has  frequently  ob- 
served cases  in  which  the  reaction  varied  from  a  strongly 
suspicious  one  to  a  plus-minus  or  negative  one  within 
periods  of  one  or  two  weeks,  while,  in  rare  instances,  a 
positive  reaction  would  be  found  negative  upon  a  second 
examination  within  the  same  period  of  time,  no  treatment 
having  been  administered  at  any  time.  In  all  such  in- 
stances subsequent  tests  resulted  in  a  positive  reaction,  so 
that  it  was  evident  that  the  variations  in  results,  provided 
the  technic  of  the  test  was  the  same,  must  have  depended 
upon  the  reduction  or  absence  of  the  body  or  bodies  in 
the  patient's  serum  which  produce  the  reaction. 

The  fact  that  the  blood  serum  of  an  undoubted  syphilitic 
patient  may,  during  certain  intervals  of  time,  give  a  nega- 
tive reaction  in  the  absence  of  specific  treatment,  and  when 
previous  and  subsequent  tests  are  positive,  is  of  practical 
importance.  While  in  the  writer's  experience  such  anom- 
alous results  have  not  been  very  numerous,  their  occur- 
rence, together  with  the  criticisms  of  the  test  by  some 
authors  who  have  had  a  similar  experience,  rendered  an 
experimental  investigation  of  the  phenomenon  of  value, 
and  in  1914,  the  writer*"  published  certain  observations 
upon  the  subject  which  will  be  briefly  discussed  at  this 
time.  The  observations  proved  that  marked  daily  varia- 
tions occur  in  the  strength  of  the  complement-fixation  re- 
action in  the  blood  of  syphilitics.  While,  a  priori,  there 
would  appear  to  be  no  reason  why  the  strength  of  the  re- 
action should  not  vary,  we  have  grown  to  consider  the 
Wassermann  reaction,  if  once  obtained,  as  a  stable  quan- 
tity, the  discordant  results  sometimes  observed  being  im- 
puted to  the  serologist  or  considered  as  proof  of  the  unre- 
liability of  the  test. 

Ten  patients  suffering  from  undoubted  syphilis  were 
selected  for  the  observations.     Most  of  these  men  were 


FACTORS   H^FLUEZ^fCING   RESULT   OF   TEST  51 

prisoners  at  the  U.  S.  Military  Prison,  at  Fort  Leaven- 
worth, and  thns  all  chance  of  the  reaction  being  influenced 
by  the  use  of  alcohol  was  obviated,  the  men  being  under  a 
rigid  discipline  and  on  a  plain,  wholesome  diet.  Of  the 
ten  patients,  two  were  in  the  primary  stage  of  the  disease, 
four  in  the  secondary  stage,  and  four  in  the  latent  stage. 
No  treatment  had  been  administered  for  over  a  year  in 
any  case,  and  then  only  to  those  who  were  in  the  latent 
stage.  The  patients  in  the  primary  stage  had  typical 
chancres  and  have  since  developed  secondary  eruptions; 
the  secondary  cases  all  presented  eruptions  and  mucous 
patches ;  while  the  four  in  the  latent  stage  were  free  from 
obvious  symptoms.  All  gave  double-plus  (four-plus)  Was- 
sermann  reactions  at  the  time  of  the  experiment. 

The  blood  was  collected  every  day  and  tested  upon  the 
same  day  it  was  collected,  daily  titrations  of  the  strength 
of  the  complement  fixation  being  made.  The  technic  used 
was  that  employed  by  the  writer  for  several  years  and 
which  is  described  in  this  work. 

Each  patient's  blood  serum  was  titrated  every  day  for 
one  week,  the  quantities  of  serum  employed  being  0.02  c.c. ; 
0.04  c.c;  0.06  c.c;  0.08  cc;  and  0.1  c.c  The  quantity  of 
blood  serum  used  with  the  technic  for  diagnostic  purposes 
is  0.1  c.c.  so  that  this  quantity  may  be  taken  as  the  diag- 
nostic amount  in  the  tables  that  follow.  A  control  of  0.1 
c.c.  of  the  serum  titrated  was  always  employed. 

In  the  tables  it  will  be  understood  that  the  sign,  +  +,  in- 
dicates absolute  inhibition  of  hemolysis,  or  a  positive  re- 
action; the  sign,  +,  anything  between  absolute  inhibition 
and  50  per  cent  of  inhibition  of  hemolysis;  the  sign,  +-, 
anything  between  50  per  cent  of  inhibition  and  total  hem- 
olysis, or  practically  a  negative  reaction;  and  the  sign,  -, 
total  hemolysis  or  a  negative  result. 

Results  in  the  Primary  Stage  of  Syphilis. — Owing  to 
lack  of  material,  only  two  cases  were  tested  in  the  primary 
stage  of  the  disease.  Both  presented  typical  chancres  and 
both  gave  a  double-plus,  or  positive,  Wassermann  reaction 


52 


THE   WASSERMANN   TEST 


when  first  tested.    Tables  V  and  VI  give  the  results  of  the 
daily  titrations  of  the  blood  serum  in  these  two  cases. 

Table  V 

Results  of  Titration  of  Blood  SefvUm  for  Complement-fixing  Strength  in 
Case  1 — Primary  Syphilis 


DATE  OF  test 

amount  of 

blood  serum  in  o.c 

's 

CONTROL. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1  C.C. 

Nov.  19 

+  - 

+ 

+ 

+ 

+ 

_ 

Nov.  20 

+  - 

+  - 

+ 

+  + 

+  + 

_ 

Nov.  21 

+  - 

+ 

+  + 

+  + 

+  + 

_ 

Nov.  22 

+  - 

+  - 

+ 

+ 

+ 

_ 

Nov.  23 

+ 

+  + 

+  + 

+  + 

+  + 

_ 

Nov.  24 

+  - 

+ 

+ 

+ 

+ 

_ 

Nov.  25 

+  + 

+  + 

+  + 

+  + 

+  + 

- 

Table  VI 

Results  of  Titration  of  Blood  Serum  for  Complement-fixing  Strength  in 
Case  2 — Primary  Syphilis 


DATE  OF  TEST 

AMOUNT  OF  BLOOD  SERUM.  C.C. 

CONTROL. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1  C.C. 

Nov.  19 
Nov.  20 
Nov.  21 
Nov.  22 
Nov.  23 
Nov.  24 
Nov.  25 

+ 
+  - 

+ 
+ 
+ 

+  -I- 

+  - 

-f-l- 
+  + 
-f-f- 

+  + 
+ 

+  - 
-f-l- 
-I-  + 
+  + 

+  + 

-n- 
-f 
-f- 

-f-4- 
+  + 
+  + 

+  + 
+  + 

+ 

+ 

+  + 
+  + 
+  + 

■" 

A  study  of  these  tables  will  show  that  considerable  varia- 
tions occurred  in  the  complement-fixing  power  of  the  blood 
serum  in  both  these  cases  of  primary  syphilis,  especially 
in  the  smaller  amounts  employed,  but  that  in  neither  case 
did  the  reaction  become  negative  with  the  diagnostic 
amount  of  serum;  i.e.,  0.1  c.c.  However,  in  one  case  a 
plus  or  doubtful  reaction  was  obtained  upon  three  of  seven 
days,  while  in  the  other  the  same  result  was  obtained  upon 
two  of  the  seven  days.  These  results  are  very  significant 
when  it  is  remembered  that  in  this  stage  of  syphilis  the 
Wassermann  test  is  so  often  doubtful,  or  negative,  and  in- 
dicates that  frequent  examinations  would  give  a  much 
higher  percentage  of  positive  results  than  are  usually  re- 


FACTOES   INFLUENCIITG   KESULT   OF   TEST 


53 


corded.  Both  of  these  cases,  if  tested  upon  certain  days 
of  the  week,  would  have  been  reported  serologically  doubt- 
ful, but,  fortunately,  in  most  primary  infections  Trepo- 
nema pallidum  may  be  demonstrated  Avith  the  dark-field, 
thus  avoiding  the  confusion  that  might  be  caused  by  varia- 
tions in  the  strength  of  the  Wassermann  reaction. 

Results   in   the    Secondary   Stage   of    Syphilis. — The 
blood  serum  of  four  typical  cases  of  secondary  syphilis, 

Table  VII 

Eesults  of  Titration  op  Serum  for  Complement-fixing  Strength  in 
Case  3 — Secondary  Syphilis 


date  of  test 

amount  of  blood  serum,  c.c. 

control  c.c. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Nov.  26 

+  + 

+  + 

+  + 

+  + 

+  + 

_ 

Nov.  27 

+  + 

+  + 

+ + 

+  + 

+  + 

_ 

Nov.  28 

+  + 

+  + 

+  + 

+  + 

+  + 

_ 

Nov.  29 

+  + 

+  + 

+  + 

+  + 

+  + 

_ 

Nov.  30 

+  + 

+  + 

+  + 

+  + 

+  + 

_ 

Dee.  1 

+  + 

+  + 

+  + 

+  + 

+  + 

_ 

Dec.  2 

+  + 

+  + 

+  + 

+  + 

+  + 

- 

Table  Vni 

Eesults  of  Titration  of  Serum  for  Complement-fesing  Strength  in 
Case  4 — Secondary  Syphilis 


DATE  OP  TEST 

amount  of  blood  serum,  c.c. 

CONTROL  C.C. 

0.02 

0.04 

0.06   1   0.08 

0.1 

0.1 

Jan.  3 
Jan.  4 
Jan.  5 
Jan.  6 
Jan.  7 
Jan.  8 
Jan.  9 

+  + 
+  + 
+  + 
+  + 

+ 

+ 

+  + 

+  + 
+  + 
+  + 

+  + 
+  + 
+  + 

+  + 
+  + 
+  + 
+  + 

+  + 
+  + 
+  + 

+  + 
+  + 

+  + 
+  + 
+  + 
+  + 

+  + 
+  + 
+  -I- 
+  + 
+  + 
+  + 
+  + 

1     1     1     1     1     1     1 

Table  IX 

Eesults  op  Titration  op  Serum  for  Complement-fixing  Strength  in 
Case  5 — Secondary  Syphilis 


date  op  test 

amount  of  blood  serum.  C.c. 

CONTROL  c.c. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Dec.  24 
Dec.  25 
Dec.  26 
Dec.  27 
Dec.  28 
Dec.  29 
Dec.  30 

+  - 
+  ~ 

+  - 

+  - 

+  - 

+ 

+  - 
+  - 

+ 

+  - 

+  - 

-f 

+  - 

+ 

+  + 

+ 
+  - 

+ 

+ 

-f + 
-f- 

+ 

+ 

+  + 
-I-  + 

- 

54 


THE    WASSEEMAN]Sr    TEST 


Table  X 

Results  of  Titration  of  Serum  for  Complement-fixixg  Strength  in 
Case  6 — Secondary  Syphilis 


amount  of  blood  serum,  c.c. 

control  c.c. 

DATE   OF  TEST 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Dec.  5 
Dec.  6 
Dec.  7 
Dec.  8 
Dec.  9 
Dec.  10 
Dec.  11 

+  + 
+  + 
+  + 
+  + 
+  - 
+  - 

+  - 
+  + 

+  + 

+  + 

+ 

+  - 

+ 

+  + 
+  + 
+  + 
+  + 

+ 

+ 

+ 

4-  + 
+  + 

+  + 
+ 
+ 

+  + 
+  + 
+  + 
+  +  . 
+  + 

+ 

+ 

- 

all  showing  lesions  of  the  disease,  was  titrated  in  the  same 
manner  as  described  and  Tables  Yll,  VIII,  IX,  and  X  give 
the  results  obtained. 

In  Case  3  the  strength  of  the  blood  sermn  did  not  vary 
at  all  during  the  time  it  was  titrated,  a  double-plus  (four- 
plus)  reaction  being  obtained,  even  with  the  smallest 
amount  of  serum  used.  The  case  is  of  interest  as  shoAving 
how  strong  the  blood  serum  is  in  complement-fixing  power 
in  some  instances  and  how  little  it  varies  in  strength  even 
when  very  small  amounts  of  serum  are  used.  The  patient 
presented  clinically  a  very  marked  secondary  eruption  over 
the  chest,  abdomen,  and  back. 

In  Case  4,  likewise,  there  was  practically  no  variation  in 
the  strength  of  the  reaction.  In  this  case  the  patient  pre- 
sented mucous  patches  in  the  mouth  and  a  well  marked 
secondary  eruption. 

In  Case  5,  the  variations  in  the  strength  of  the  comple- 
ment-fixing power  of  the  blood  serum  varied  marked^, 
especially  when  amounts  of  serum  of  less  than  0.1  c.c.  were 
used.  With  the  diagnostic  amount  of  serum;  i.e.,  0.1  c.c, 
the  serum  gave  a  plus-minus,  or  practically  negative  result 
upon  one  day  and  a  plus,  or  doubtful  reaction  upon  two  of 
the  seven  days  during  which  it  was  tested.  With  0.08  c.c. 
of  the  serum,  an  amount  which  was  formerly  used  in  the 
writer's  laboratory  as  the  diagnostic  amount,  a  negative 
reaction  was  obtained  upon  one  day  and  plus-minus  upon 
one  day  and  a  plus,  or  doubtful  reaction,  upon  two  days. 


FACTORS   INFLUENCIls^G   RESULT    OF   TEST 


55 


In  this  case  the  Wassermann  reaction  was  practically  nega- 
tive on  one  da}^  and  doubtful  upon  two  days,  although  very 
marked  secondary  symptoms  were  present. 

In  Case  6  the  variation  in  the  strength  of  the  reaction 
was  not  very  marked,  when  the  diagnostic  amount  of  blood 
serum  was  used,  the  serum  giving  absolute  inhibition  of 
hemolysis  upon  all  but  two  days,  when  it  gave  a  plus  or 
doubtful  reaction. 

The  results  of  the  titration  of  the  blood  serum  in  second- 
ary cases  demonstrate  that  variations  do  occur  daily  in 
cases  showing  well  marked  symptoms  of  the  disease  and 
that  a  case  giving  absolute  inhibition  of  hemolysis  upon 
one  day  may  be  negative  or  doubtful  upon  a  succeeding 
day.  When  small  amounts  of  blood  serum  are  used  the 
variations  in  the  strength  of  the  reaction  are  very  marked, 
in  some  instances,  as  in  Case  5,  while  in  others  the  strength 
of  the  reaction  remains  unchanged.     (Case  3.) 

Eesults  in  the  Lateistt  Stage  of  Syphilis. — Four  pa- 
tients in  the  latent  stage  of  syphilis  were  tested,  all  of 
whom  gave  a  history  of  definite  s\Tiiptoms  of  the  disease, 
but  in  none  had  symptoms  been  present  mthin  one  year. 
All  of  the  men  had  received  one  or  two  injections  of  sal- 
varsan  and  all  gave  a  double-plus  (four-plus)  Wassermann 
reaction  shortly  before  the  titrations  of  the  blood  serum 
were  commenced.  Tables  XI,  XII,  XIII,  and  XIV  give  the 
results  of  the  titrations. 

Table  XI 

Results  of  Titration  of  Serum  for  Complement-fixing  Strength  in 
Case  7 — Latent  Syphilis 


date  of  test 

AMOUNT  OP  blood  SERUM.  C.C. 

CONTROL  C.C. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Dec.  19 
Dec.  20 
Dec.  21 
Dec.  22 
Dec.  2.3 
Dec.  24 
Dec.  25 

+  - 

+  + 
+  - 

+ 
+  - 

+ 
+  - 
+  + 
+  + 

+ 

+ 

+ 
+  + 

+ 
+  + 

+ 

+  + 

+ 
+  + 

+ 

+  + 
+  + 

+ 

+  + 

+ 
+  + 

+ 

+  + 
+  + 

+ 

- 

56 


THE   WASSERMANN   TEST 


Table  XII 

Eesults  of  Titration  of  Serum  for  Complement-fixing  Strength  in 
Case  8 — Latent  Syphilis 


amount  of  blood  serum,  c.c. 

control  c.c. 

DATE  OF  TEST 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Jan.  12 
Jan.  13 
Jan.  14 
Jan.  15 
Jan.  16 
Jan.  17 
Jan.  18 

+ 

+ 

+ 

+  - 
+  - 
+  - 
+  - 

+  + 

+  + 

+  + 

+ 

+ 
+ 
+ 

+  + 
+  + 
+  + 

+  + 
+  + 
+  + 

+ 

+  + 
+  + 
+  + 

+  + 
+  + 

+ 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 

; 

Table  XIII 

Results  of  Titration  of  Serum  for  Complement-fizing  Strength  in 
Case  9 — Latent  Syphilis 


amount  op  blood  serum,  c.c. 

CONTROL  C.C. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Jan.  15 
Jan.  16 
Jan.  17 
Jan.  18 
Jan.  19 
Jan.  20 
Jan.  21 

+  - 

+  - 

+  - 

+ 

+  + 

+ 

+ 
+  - 

+  - 

+  + 

+ 

+  + 

+ 

+ 
+  - 

+  + 
+ 

+  + 
+  + 

+  + 
+  - 

- 

Table  XIV 

Results  of  Titration  of  Serum  for  Complement-fixing  Strength  in 
Case  10 — Latent  Syphilis 


amount  of  blood  serum.  C.C. 

CONTROL  C.C. 

0.02 

0.04 

0.06 

0.08 

0.1 

0.1 

Jan.  15 
Jan.  16 
Jan.  17 
Jan.  18 
Jan.  19 
Jaji.  20 
Jan.  21 

-f- 

+  - 

+  - 

+  - 

-f- 

+  - 
+  - 
+  - 
+  -(- 

+  - 

-l- 

+ 

-1- 
-f- 

+ 
-f-l- 

-f 

+ 

+ 

-f- 

- 

In  Case  7  the  strength  of  the  complement-fixation  re- 
action, using  the  diagnostic  amount  of  serum,  i.  e.,  0.1  c.c, 
was  only  sufficient  upon  three  of  the  seven  days  that  the 
serum  was  tested  to  give  a  plus  or  doubtful  reaction,  while 
with  smaller  amounts  the  variations  in  the  strength  were 
very  marked.  Upon  these  three  days  the  test  would  have 
been  reported  as  doubtful  and  if  further  tests  had  not  been 


FACTORS   lE'FLUENCII^G   RESULT    OF   TEST  57 

made,  the  diagnosis,  owing  to  the  absence  of  symptoms, 
would  have  been  missed. 

In  Case  8  there  was  no  variation  from  a  positive  reaction 
with  the  diagnostic  amonnt  of  sernm  during  the  seven  days 
the  titrations  were  made,  although  some  variation  occurred 
with  smaller  amounts  of  the  serum. 

In  Case  9  there  were  very  marked  variations  in  the 
strength  of  the  reaction  with  the  diagnostic  amount  of  se- 
rum, a  negative  reaction  being  obtained  upon  one  day,  a 
plus-minus  upon  another,  and  a  plus  or  doubtful  reaction 
upon  a  third.  A  double-plus,  or  positive  reaction,  was  ob- 
tained upon  only  four  of  the  seven  days  that  the  serum 
was  tested.  With  amounts  of  serum  smaller  than  the  diag- 
nostic amount  of  0.1  c.c.  the  variations  were  very  great. 

In  Case  10  there  was  also  great  variation,  from  day  to 
day,  in  the  strength  of  the  complement-fixation  reaction,  the 
diagnostic  amount  of  serum  giving  a  plus-minus  reaction 
upon  one  day,  a  plus  reaction  upon  three  days,  and  a  double- 
plus  or  positive  reaction  upon  three  days  of  the  week.  "With 
smaller  amounts  of  serum  the  variations  were  still  more 
marked. 

GEiiTERAL  Discussion. — The  most  important  practical  point 
brought  out  by  these  observations  on  the  daily  titration  of 
the  blood  serum  of  untreated  syphilitics  is  that  great  varia- 
tions may  occur  in  the  complement-binding  x^ower  of  the  se- 
rum of  patients  uninfluenced  by  any  kind  of  treatment,  and 
that  these  variations  occur  from  day  to  day,  so  that  one  neg- 
ative examination,  or  even  more,  in  a  suspected  case,  is  of 
absolutely  no  value  in  excluding  the  disease.  In  several 
of  the  cases  mentioned  the  blood  serum,  if  tested  upon  cer- 
tain days  only,  would  have  given  a  negative,  or  practically 
negative,  result,  although  serum  from  the  same  case  had 
been  previously  positive,  and  again  became  positive  within 
a  day  or  two.  If  this  be  true  of  untreated  cases,  some  of 
them  showing  severe  lesions  of  the  disease,  it  will  certainly 
be  found  true  of  a  greater  number  of  latent  and  treated  in- 
fections, the  class  of  cases  in  which  a  negative  Wassermann 


58  THE   WASSERMANN   TEST 

reaction  is  so  often  considered  decisive  as  to  the  absence 
of  the  disease.  The  results  of  these  tests  indicate  the  great 
value  of  repeated  examinations,  when  a  negative  Wasser- 
mann  is  reported,  before  a  patient  is  considered  as  cured, 
or  before  he  is  assured  that  he  is  not  suffering  from  a 
syphilitic  infection. 

As  to  the  cause  of  the  variations  in  the  strength  of  the 
reaction  observed  from  day  to  day,  it  is  evident  that  the 
antibodies  and  lipotropic  substances  present  in  the  pa- 
tient's serum  must  vslyy  from  day  to  day,  or  such  results 
would  not  be  obtained.  It  may  be  accepted  as  proved  that 
there  exist  in  the  blood  of  syphilitics  antibodies  and  lipo- 
tropic substances  that  are  capable  of  fixing  complement 
in  the  presence  of  a  proper  antigenic  extract,  and  that  these 
bodies  depend  for  their  origin  on  the  presence  in  the  body 
of  living  treponemas,  for  when  the  latter  disappear  from 
the  lesions  in  experimental  animals,  as  the  result  of  treat- 
ment, the  complement-fixation  reaction  also  disappears. 
AAHiether  these  substances  are  produced  by  the  reaction  of 
the  tissues  to  endotoxins  liberated  by  the  breaking  down 
of  dead  treponemas ;  to  toxins  produced  by  the  living  trep- 
onemas ;  or  to  the  presence  in  the  blood  of  the  toxins  them- 
selves; are  all  mooted  questions,  but  these  experiments 
prove  that  there  must  be  a  certain  amount  of  them  in  the 
blood  serum  before  a  positive  Wassermann  reaction  can 
be  obtained,  and  that  this  amount  varies  considerably  from 
day  to  day. 

It  is  but  just  to  call  attention  to  the  importance  of  these 
results  in  explaining,  possibly,  the  discrepancies  between 
Wassermann  reports  from  various  laboratories  where  spec- 
imens of  blood  from  the  same  individual  were  examined  at 
different  times,  for  it  is  evident  that  unless  the  same  spec- 
imen of  blood  be  examined  no  reliable  conclusions  can  be 
draMTi  regarding  the  reports  of  different  laboratories,  a 
fact  that  should  be  borne  in  mind  when  it  is  desired  to 
obtain  a  report  from  more  than  one  laboratory  upon  a 
suspected  individual. 


FACTOES    INFLUElSrCING   KESULT    OF    TEST  59 

The  factors  which  have  been  mentioned  in  this  chapter 
as  markedly  influencing  the  results  of  the  Wassermann 
test ;  i.  e.,  the  effect  of  alcohol ;  of  bacteria  growing  in  the 
blood  serum  to  be  tested;  and  normal  variations  in  the 
power  of  the  blood  serum  to  bind  complement,  are  all  of 
practical  importance  and  should  all  be  considered  in  per- 
forming the  test  and  in  judging  of  its  results  in  any  case 
where  there  is  a  question  of  the  accuracy  of  the  reaction. 
If  this  were  done,  there  would  be  much  less  criticism  of 
the  Wassermann  test  than  there  is,  and  a  much  better  un- 
derstanding of  its  reliability  and  its  limitations. 


CHAPTER  IV 

PREPARATION  AND  TITRATION  OF  THE 

REAGENTS  USED  IN  THE 

WASSERMANN  TEST 

The  following  list  of  apparatus  and  reagents  needed  in 
performing  the  Wassermann  test  is  here  inserted  and  it 
will  be  found  to  include  all  that  is  really  essential  for  this 
work: 

List  of  Apparatus 

1  Luer  syi-inge,  capacity  1  c.c. 
1  Luer  syiinge,  capacity  5  c.c. 
1  Luer  syringe,  capacity  20  c.c. 
6  needles,  for  collecting  blood,   (special  needles  for  this  purpose 

can  be  obtained  of  any  good  surgical  supply  house). 
1  electric  centrifuge,  International  Instrument  Co.,  Size  1,  Type 
A,  with  one  four  15  c.c.  tube  head  and  metal  holders,  and 
one  four  50  c.c.  tube  head  and  metal  holders.     Type  of 
current  and  voltage  must  be  specified. 
20  centrifuge  tubes,  plain,  15  c.c. 
10  centrifuge  tubes,  graduated,   15   c.c. 
10  centrifuge  tubes,  50    c.c,   plain. 
1  incubator,  either  electric,  gas,  or  oil,  with  proper  thermoregu- 

lators,  to  run  at  56°  C. 
1  water-bath,    with    thermometer,    burner    and    thermoregulator, 
large    enough    to    accommodate    the    required    number    of 
Wassermann  racks,  to  run  at  37°  C. 
1  set  of   20   copper   test   tube  racks,  made   by  Topham,  Wash- 
ington, D.  C. 
1  amboceptor  cutter,  ruade  by  Topham,  Wasliington,  D.  C. 
1  set  of  3  amboceptor  markers,  4  mm.,  5  mm.,  and  6  mm.,  made 

by  Topham,  Washington,  D.  C. 
1  paraffin  bath  to  fit  incubator  mentioned  above,  to  hold  tubes 
during   inactivation  of  blood  serum. 
100  or  more  test  tubes,  without  lip,  100  by  12.5  millimeters. 
4  test  tube  baskets,  6  inches  high  by  7.5  inches  in  diameter. 
10  Mohr  pipettes,  10  c.c.  capacity,  graduated  in  tenths. 
10  Mohr  pipettes,     5  c.c.  capacity,  graduated  in  tenths. 

60 


PREPAKATIOX   AND   TITRATION   OF   REAGENTS  61 

20  pipettes,  serologic,  1  c.c.  capacity,  graduated  in  hundredths  to 
tip.  These  pipettes  should  be  of  small  bore  and  the 
graduations  far  enough  apart  to  render  accurate  read- 
ings possible  for  quantities  as  small  as  one-one-hundredth 
of  a  c.c. 
100  or  more  pipettes,  serologic,  capacity  1  c.c,  graduated  in  tenths. 
These  are  used  for  measuring  the  sera  to  be  tested. 
12  Erlenmeyer  flasks,  capacity  250  c.c.  each. 

6  animal  cages. 
20  pounds  soft  paraffin,  melting  point  43°   C. 
1  bottle,  500  gm.  sodium  chloride,  C.  P. 
1  bottle,  30  gm.  cholesterin,  Merck's. 
1  bottle,  500  gm.  absolute  alcohol. 
6  triangular  tiles,  3  inches  long. 

Guinea  pigs  and  ralibits  must  be  kept  for  complement 
and  amboceptor  production  and  a  small  and  suitable  room 
or  animal  house  should  be  set  aside  to  shelter  them  and  to 
allow  for  breeding. 

Preparation  of  Reagents 

The  proper  preparation  of  the  reagents  used  in  the  Was- 
sermann  test  is  of  great  importance  and  explicit  directions 
are  given  in  the  following  pages  regarding  this  subject, 
which,  if  carefully  adhered  to,  will  result  in  the  prepara- 
tion of  reagents  that  will  give  accurate  results. 

The  Wassermann  test  as  performed  in  the  writer's  lab- 
oratory and  in  most  of  the  laboratories  connected  with  the 
Medical  Department  of  the  Army,  is  a  modification  of  both 
the  test  as  originally  recommended  b^^  Wassermann,  and 
the  modification  of  that  test  recoimnended  by  Noguchi.  The 
technic  was  originally  worked  out  b}^  the  writer  and  has 
since  been  standardized,  so  far  as  the  apparatus  is  con- 
cerned, by  Lieut.  Colonel  Vedder,  of  the  Medical  Corps. 
A  human  hemolytic  s^^stem  is  employed  instead  of  the 
sheep  system  recommended  by  Wassermann;  alcoholic  ex- 
tracts of  both  foetal  syphilitic  liver,  and  cholesterinized 
alcoholic  extracts  of  normal  human  heart  muscle,  are  used 
as  antigen ;  and  the  blood  serum  is  inactivated  at  56°  C.  for 


62  THE    WASSEKMAXIir    TEST 

one-half  hour  before  it  is  tested.    The  follo^^ing  reagents 
are  employed  in  the  test : 

1.  Complement.    The  fresh  blood  serimi  of  guinea  pigs. 

2.  Hemolytic  amboceptor.  The  blood  sermn  of  rabbits 
immunized  to  human  red  blood  corpuscles. 

3.  Hemolytic  antigen.  A  suspension  of  human  red  blood 
corpuscles. 

4.  Syphilitic  antigen.  An  alcoholic  extract  of  foetal 
S3T3hilitic  liver  and  a  cholesterinized  alcoholic  extract  of 
normal  human  heart  muscle. 

5.  Syphilitic  amboceptor.  The  patient's  blood  serum. 
This  may  or  may  not  contain  the  lipotropic  substance  or 
substances  necessary  for  complement  fixation  "vvith  the 
antigens  mentioned. 

Preparation  of  Complement. — Complement  is  a  constit- 
uent of  the  fresh  blood  serum  of  all  animals  but  certain  an- 
imals furnish  better  complement  for  complement-tixation 
tests  and  for  the  Wassermann  test  than  do  others,  and  it 
has  been  demonstrated  that  for  the  Wassermann  test  the 
blood  serum  of  the  guinea  pig  supplies  a  better  complement 
than  does  that  of  other  animals.  Noguchi  and  Bronfen- 
brenner  tested  the  blood  serum  of  the  sheep,  dog,  ox,  guinea 
pig,  hog,  and  rabbit,  and  found  that  the  serum  of  the  guinea 
pig  gave  the  best  results  when  used  as  complement  in  the 
Wassermann  test,  so  that  the  blood  serum  of  this  animal 
has  been  generally  adopted  for  this  purpose. 

The  blood  serum  of  different  guinea  pigs  varies  consid- 
erably in  complementary  activity,  even  in  health,  and  in 
unhealthy  animals,  and  when  the  temperature  is  either 
very  hot  or  cold,  marked  variations  may  occur.  For  this 
reason  it  is  essential  that  the  blood  serum  be  titrated  for 
its  complementary  power  each  time  that  it  is  used,  and  it 
is  always  best,  in  practice,  to  employ  the  serum  from  two 
or  three  guinea  pigs,  mixed,  rather  than  to  depend  upon 
that  of  a  single  pig,  which  might  be  found  to  be  so  weak  as 
to  be  inconvenient  for  use  in  the  test. 


Fig.  3. 
Luer  syringes.      Set  of  seven,   suitable  for  use   in  the   Wassermann  test,   and   for   serologic 

work  in  general. 


PREPARATION   AND   TITRATION    OF   REAGENTS  63 

The  guinea  pig  may  be  either  bled  from  the  heart  or  its 
throat  cut.  It  is  the  practice,  in  the  writer's  laboratory, 
to  bleed  the  pigs  from  the  heart,  as  by  this  method  the 
animal  is  not  sacrificed  and  can  be  used  over  and  over  again 
for  bleeding,  provided  the  bleedings  be  from  three  to  four 
weeks  apart.  Several  lots  of  pigs  can  be  rotated  and  thus 
considerable  expense  saved,  as  the  animals  usually  stand 
the  bleeding  well  and  it  is  seldom  that  any  are  lost  if  the 
bleeding  is  properly  done.  The  operation  should  be  per- 
formed under  ether  and  consists  simply  in  entering  the 
heart  with  a  hypodermic  needle  attached  to  a  syringe  hold- 
ing 10  c.c.  and  nearly  this  amount  of  blood  can  be  aspi- 
rated from  the  heart  without  danger  to  the  animal.  It 
is  well  to  connect  the  needle  of  the  syringe  to  the  syringe 
with  a  flexible  rubber  tube,  as  it  is  much  easier  to  make  the 
puncture  and  handle  the  syringe  afterward  if  this  is  done. 
The  operation  requires  considerable  practice  but  when  the 
technic  is  once  mastered  it  is  by  far  the  most  satisfactory 
way  of  obtaining  complement  for  the  Wassermann  test, 
especiall}^  if  only  a  small  number  of  tests  are  to  be  made 
at  one  time. 

After  bleeding,  the  blood  should  be  ejected  from  the 
syringe  into  a  sterile  Petrie  dish,  allowed  to  stand  at  room 
temperature  for  two  hours  and  then  placed  in  the  ice  box 
overnight.  In  the  morning  the  clear  serum  is  pipetted  off 
and  if  it  is  to  be  used  at  once,  diluted  with  an  equal  amount 
of  normal  salt  solution — (0.85%).  If  it  is  not  to  be  used 
for  several  hours  it  should  be  placed  at  once  in  the  ice  box, 
as  the  complementary  power  of  blood  serum  is  weakened 
by  exposure  to  room  temperature  for  any  great  length  of 
time.  The  serum  should  be  free  from  blood  corpuscles 
and  almost  colorless  or  but  very  slightly  blood  stained. 

If  a  very  large  number  of  tests  are  to  be  made  it  may 
be  better  to  sacrifice  the  pigs  by  cutting  their  throats,  each 
pig  being  held  over  a  Petrie  dish,  the  throat  cut  quickly 
with  a  sharp  scalpel  or  razor,  after  shaving  the  neck,  and 
the  blood  allowed  to  run  into  the  dish.    Great  care  should 


64  THE   WASSEEMANN   TEST 

be  taken  not  to  cut  too  deeply,  so  as  to  penetrate  the  esoph- 
agus or  trachea,  as  the  secretions  from  the  stomach  have 
a  marked  inhibitory  effect  upon  complement  and  this  is 
also  true  to  a  lesser  degree  of  the  secretions  of  the  larynx 
or  trachea.  After  collecting,  the  blood  should  be  treated 
in  the  same  manner  as  after  puncture  of  the  heart. 

The  syringe,  needles,  Petrie  dishes,  and  all  apparatus 
used  in  collecting  the  blood  for  complement  should  be  ster- 
ile and  perfectly  dry,  AAdth  the  exception  of  the  S3^ringe, 
which  should  be  washed  out  with  normal  salt  solution  be- 
fore the  puncture  is  made.  If  the  blood  serum,  after  be- 
ing pipetted  off,  is  much  blood  stained,  it  should  not  be 
used  for  complement.  If  it  contains  blood  corpuscles  these 
may  be  removed  by  centrifuging  and  pipetting  off  the  clear 
serum  above  the  layer  of  corpuscles  at  the  bottom  of  the 
centrifuge  tube. 

The  complement,  even  after  being  diluted  one-half  with 
normal  salt  solution,  may  be  kept  in  the  ice  box  without 
much  loss  of  strength  for  about  three  days,  but  usually 
upon  the  third  day  the  titration  of  it  will  show  such  loss 
of  complementary  power  that  it  will  be  useless  for  the 
test.  Complement  appears  to  be  at  the  height  of  its  com- 
plementary power  about  thirty-six  hours  after  removal 
from  the  pig,  but  is  much  weaker  immediately  after  with- 
drawal of  the  blood  and  grows  gradually  weaker  from  day 
to  day  after  this  period. 

Preservation  of  Complement. — Many  attempts  have  been 
made  to  preserve  complement  by  the  addition  of  various 
substances,  but  none  of  the  methods  so  far  advocated  have 
been  generally  adopted.  Noguchi*^  attempted  to  preserve 
it  by  impregnating  filter  paper  with  it,  a  method  very  suc- 
cessful with  amboceptor  serum,  but  without  satisfactory 
results.  Kolmer*^  recommends  chemically  pure  sodium 
chloride  as  the  best  preservative,  adding  0.425  gram  so- 
dium chloride  to  each  10  c.c.  of  sera  obtained  from  several 
guinea  pigs.    He  states  that  complement  preserved  in  this 


PREPAKATION   AND   TITRATION   OF   REAGENTS  65 

manner  will  maintain  its  hemolytic  and  fixing  properties 
for  several  weeks. 

Preparation  of  Hemolytic  Amboceptor.— The  hemolytic 
serum  or  amboceptor  nsed  in  the  Wassermann  test  as  per- 
formed by  the  writer  is  prepared  by  immunizing  rabbits 
to  human  red  blood  corpuscles.  Noguchi*^  was  the  first  to 
call  to  the  attention  of  serologists  the  advantage  of  a  hu- 
man hemolytic  system  in  Wassermann  work  and  the  writer 
believes  that  it  is  the  system  best  suited  for  complement- 
fixation  reactions  upon  the  blood  of  man.  Its  adoption  ob- 
viates the  danger  that  is  present  in  human  blood  serum,  of 
the  action  of  the  natural  antisheep  amboceptor  normally 
present  in  such  blood  serum,  and  which,  when  the  sheep 
hemolytic  system  is  employed,  should  be  removed  from 
such  serum  before  the  test  is  made.  It  has  not  been  prac- 
ticable, owing  to  the  immense  number  of  Wassermann  tests 
made  in  army  laboratories,  to  use  any  method  which  re- 
moves this  natural  antisheep  amboceptor  from  blood  se- 
rum, and,  therefore,  aside  from  the  superiority  of  the 
human  hemolytic  system  for  complement-fixation  work,  it 
was  necessary  to  adopt  it  from  practical  considerations. 
The  only  objection  that  can  be  urged  against  the  human 
system  is  that  it  is  more  difficult  to  immunize  rabbits 
against  human  erythrocytes  than  against  sheep  corpuscles 
and  that  it  requires  a  more  careful  technic  both  in  collect- 
ing the  blood  and  injecting  it  into  the  animals,  but  this 
objection  is  of  no  weight  when  the  great  advantages  of  the 
human  hemolytic  system  are  considered. 

Before  considering  the  method  of  preparing  the  hemo- 
lytic amboceptor  serum  there  are  certain  general  principles 
regarding  amboceptors,  either  bacteriolytic  or  hemolytic, 
that  will  be  mentioned. 

It  will  be  remembered  that  amboceptors  are  produced  in 
the  blood  serum  of  an  animal  in  response  to  the  injection 
of  an  antigen,  and  that  these  amboceptors  are  specific  for 
the  antigen  injected.  If  the  blood  serum  of  the  animal  con- 
taining them  is  added  to  a  mixture  of  the  antigen  and  of 


66  THE   WASSEKMAE-N   TEST 

fresh  blood  serum,  or  complement,  the  amboceptor  will  act 
as  an  intermediary  body,  allowing  the  complement  to  act 
upon  the  antigen,  and  bacteriolysis  or  hemolysis  will  result. 

Amboceptors  are  thermostable ;  i.  e.,  their  activity  is  not 
destroyed  by  heating  the  serum  containing  them  for  one 
half  hour  at  56°  C.  They  are  also  resistant  to  a  consider- 
able degree  to  acids  and  alkalies,  and  to  drying.  A  good 
hemolytic  serum  can  be  preserved  upon  filter  paper,  in  a 
dry  condition,  for  many  months  with  very  little  loss  of 
strength  and  this  fact  is  made  use  of  in  the  Wassermann 
test,  as  will  be  noted  later. 

Amboceptors  appear  to  act  as  a  chemical  linking  body, 
connecting  the  antigen  with  the  complement,  as  the  comple- 
ment is  unable  to  act  upon  an  antigen  except  through  the 
agency  of  the  amboceptor.  Hemolytic  amboceptors  occur 
in  very  small  amounts  in  the  blood  of  normal  animals  and 
such  amboceptors  are  sometimes  present  in  an  amount  suf- 
ficient to  cause  trouble  in  complement-fixation  reactions,  as 
in  the  case  of  the  normal  antisheep  amboceptor  present 
in  human  blood  serum. 

In  producing  hemolytic  amboceptor  for  human  red  blood 
corpuscles  in  rabbits  several  methods  have  been  recom- 
mended which  "will  be  described.  The  writer  has  obtained 
the  best  results  with  the  method  first  described  but  the 
other  methods  are  given  in  order  that  a  choice  may  be 
made  if  desired,  according  to  circumstances. 

Well  grown,  perfectly  healthy  rabbits  are  selected,  pref- 
erably pure  white  ones,  and  are  given  repeated  injections 
of  washed  human  red  blood  corpuscles.  The  blood  corpus- 
cles are  prepared  for  the  injections  in  the  following  man- 
ner :  The  requisite  amount  of  blood,  allowing  at  least  twice 
the  amount  of  blood  as  the  amount  of  corpuscles  to  be  in- 
jected, is  withdrawn  from  one  of  the  large  veins  of  the 
arm  with  a  glass  syringe,  which  has  been  sterilized  and 
washed  out  with  sodium  citrate  solution.  After  drawing 
the  blood,  it  is  at  once  ejected  into  a  flask  containing  from 
200  to  250  c.c.  of  normal  salt  solution  (0.85%),  and  dis- 


Fig.   4. 
Klectric    centrifuge. 


PREPABATION   AISTD   TITRATION   OF   REAGENTS  67 

tributed  into  large  centrifuge  tubes  (50  c.c.)  and  centri- 
fuged  until  the  erythrocytes  are  deposited  and  the  super- 
natant fluid  is  practically  colorless.  The  supernatant  fluid 
is  now  poured  or  pipetted  off  and  the  tubes  again  filled 
with  the  salt  solution,  well  shaken  so  as  to  distribute  the 
red  cells  throughout  the  solution,  and  again  centrifuged. 
This  process  is  repeated  four  times,  when  the  supernatant 
salt  solution  is  tested  for  albumin;  if  the  merest  trace  is 
demonstrated,  the  washing  is  again  repeated  and  continued 
until  no  reaction  for  albumin  is  obtained.  Almost  invari- 
ably four  washings  will  be  found  sufficient.  Care  should 
be  taken  not  to  centrifuge  for  so  long  that  the  corpuscles 
become  packed  at  the  bottom  of  the  tubes,  for  the  force 
necessary  to  dislodge  them  will  break  up  a  certain  per- 
centage and  render  them  unsuitable  for  injection.  Care 
should  also  be  taken  to  have  each  centrifuge  tube  weigh 
the  same  as  all  others,  and  this  can  be  done  by  weighing 
each  in  a  balance  at  the  time  they  are  filled.  Unless  this 
is  done  the  centrifuge  will  run  unevenly  and  is  liable  to 
breakage  or  accident. 

After  the  corpuscles  are  washed  in  the  manner  described 
they  are  mixed  with  as  little  normal  salt  solution  (0.85) 
which  must  be  sterile,  as  is  necessary  to  secure  a  suspen- 
sion that  will  pass  through  the  needle  of  the  injection  syr- 
inge, and  are  at  once  injected,  either  intravenously  or 
subcutaneously,  with  a  sterilized  syringe. 

Methods  of  Securing  a  Good  Amboceptor  Serum. — The 
methods  of  securing  a  good  amboceptor  serum  vary  some- 
what but  the  writer  has  found  that  the  most  rapid  method 
of  immunizing  the  rabbits  and  securing  a  good  hemolytic 
serum  is  to  give  an  intravenous  injection,  in  the  ear  vein, 
of  1  c.c.  of  washed  erythrocytes  every  other  day  until  five 
or  six  injections  have  been  given,  when  the  rabbit's  blood 
serum  should  be  titrated  and  will  generally  be  found  strong 
enough  in  hemolytic  properties  to  be  used  for  the  test. 
If  it  is  not,  two  or  three  more  intravenous  injections  of  the 
same  amount  of  red  corpuscles  should  be  given.    Rarely, 


68  THE   WASSEKMANN   TEST 

animals  are  found  that  will  not  develop  a  sufficiently  strong 
serum,  no  matter  liow  many  injections  may  be  given,  but 
usually  from  five  to  six  injections  will  be  found  sufficient. 
By  this  method  it  is  very  seldom  that  an  animal  is  lost, 
as  the  frequent  injections  prevent  the  development  of  ana- 
phylaxis, and  a  good  serum  for  use  as  amboceptor  in  the 
Wassermann  test  can  be  secured  within  a  comparatively 
short  period  of  time. 

Another  good  method  is  to  give  one  subcutaneous 
injection  of  5  c.c.  of  washed  human  erythrocytes  in  the 
abdomen,  followed  by  from  three  to  four  intravenous  in- 
jections in  the  marginal  vein  of  the  ear  of  from  3  to  4  c.c. 
of  washed  erythrocytes,  at  intervals  of  from  five  to  six 
days.  This  method  is  much  slower  than  the  preceding,  and 
not  infrequently  an  animal  is  lost  through  anaphylactic 
phenomena. 

Noguchi**  recommends  the  following  method  of  produc- 
ing hemolytic  amboceptor  for  the  Wassermann  test:  Five 
injections  of  washed  human  erythrocytes  are  given  subcu- 
taneously  in  the  abdomen,  the  first  of  5  c.c. ;  the  second,  8 
c.c. ;  the  third,  12  c.c. ;  the  fourth,  15  c.c. ;  and  the  fifth,  20 
c.c;  at  five  day  intervals.  Nine  days  after  the  last  injec- 
tion the  rabbit  is  bled  for  a  small  amount  of  blood  from  the 
ear  vein  and  the  serum  titrated.  This  method  is  an  excel- 
lent one  but  consumes  much  more  time  than  the  method  of 
intravenous  injections  made  every  other  day,  recommended 
above. 

Whatever  method  is  used  for  immunizing  the  rabbits,  the 
blood  serum  should  not  be  tested  for  its  hemolytic  proper- 
ties until  from  7  to  9  days  have  elapsed  from  the  date  of 
the  last  injection  of  red  corpuscles. 

Preservation  of  the  Amboceptor  Serum. — The  ambocep- 
tor serum  may  be  preserved  in  ampules  holding  from  1 
to  5  c.c.  in  the  ice  box,  and  used  as  required,  but  the  writer 
has  employed  for  nearly  ten  years  the  method  of  preserv- 
ing amboceptor  serum  for  the  Wassermann  test  recom- 
mended by  Noguchi  with  the  most  satisfactory  results  and 


PREPARATION   AND   TITRATION   OF   REAGENTS 


69 


this  method  is  used  in  most  of  the  army  laboratories.  It 
consists  in  impregnating  suitable  filter  paper  with  the 
blood  serum,  drying,  and  storing  in  a  glass  container  in 
a  dark  closet  or  drawer.  The  method  has  entirely  replaced 
the  use  of  liquid  amboceptor  in  our  laboratories  and  pre- 
served in  this  manner  the  serum  keeps  much  better,  and 
preserves  its  strength  for  a  much  longer  time,  than  when 
it  is  kept  in  liquid  form,  and  the  danger  of  bacterial  con- 
tamination, always  present  with  the  liquid  serum,  is 
avoided. 

Before  impregnating  the  filter  paper,  a  preliminary  ti- 
tration of  the  rabbit's  blood  serum  should  be  made  in  order 
to  determine  whether  it  is  strong  enough  in  hemolytic 
amboceptor  to  use  in  the  test.  For  this  purpose  a  small 
amount  of  the  rabbit's  blood  is  collected  from  the  ear  vein 
in  a  capillary  pipette  or  Wright  tube  and  the  serum  allowed 
to  separate.  It  is  then  separated  from  the  clot  and  inac- 
tivated by  heating  it  for  one-half  hour  at  56°  C.  in  the 
paraffin  bath  or  water-bath,  and  one  drop  of  it,  from  a 
capillary  pipette,  is  mixed  with  39  drops  of  sterile  normal 
salt  solution  (0.85)  and  titrated  for  hemolytic  strength. 
The  method  of  titration  is  illustrated  in  Table  XV. 

Table  XV 
Preliminary  Titration  op  Amboceptor  Serum  for  Wassermann  Test* 


TUBE 

no. 


AMOUNT       OF 
SALT       SOLU- 
TION. 0.85%. 
G.C. 


0.9 
0.9 
0.9 
0.9 
0.9 
0.9 


NUMBER  OF 

COMPLEMENT 

UNITS 


AMOUNT    OF 
BLOOD  SUS- 
PENSION. 

5%. 
c.c. 


0.1 
0.1 
0.1 
0.1 
0.1 
0.1 


AMOUNT  OF 
AMBOCEPTOR 
SERUM,  DI- 
LUTED 1-40. 
DROPS 


1 

2 
3 
4 
5 

None 


Incubate  i  n 
water-bath  at 
-37°  C.  for  one 
hour  or  in  incu- 
bator at  same 
temperature  for 
two  hours  and 
read  results. 


*If  the  unit  of  complement  has  not  been  determined  by  a  previous  titration,  use  0.05 
c.c.  of  a  1 :2  dilution  in  salt  solution. 


After  incubating  in  the  water-bath  at  37°   C.  for  one 
hour,  or  in  the  incubator  for  two  hours,  the  titration  is 


70  THE   WASSEKMANN   TEST 

read,  and  if  either  of  tlie  tubes  containing  one  or  two  drops 
of  the  rabbit's  sernm  shows  complete  hemolysis,  the  serum 
is  strong  enough  in  hemolytic  amboceptor  for  use  in  the 
test  and  the  rabbit  should  be  bled  at  once.  The  control 
tube,  No,  6,  should  show  complete  inhibition  of  hemolysis. 

Bleeding  is  accomplished  most  quickly  by  cutting  the 
carotid  vessels  while  holding  the  rabbit  over  a  large  glass 
dish,  into  which  the  blood  is  allowed  to  flow.  The  neck  of 
the  animal,  where  the  incision  is  to  be  made,  should  be 
shaved  and  washed  carefully  but  no  disinfectant  should  be 
used.  The  glass  dish  to  contain  the  blood  should  be  ster- 
ilized before  use  and  after  the  blood  has  been  collected  it 
should  be  kept  at  room  temperature  for  about  two  hours 
and  then  placed  in  the  ice  box  overnight.  In  the  morning 
the  clear  serum  should  be  pipetted  off  into  small  test  tubes, 
inactivated  by  heating  it  for  one-half  hour  at  56°  C.  in  the 
paraffin  or  water-bath,  and  then  kept  in  the  ice  box  until 
one  is  ready  to  impregnate  the  filter  paper.  It  does  no 
harm  if  the  blood  serum  is  slightly  tinged  Avith  hemoglobin, 
as  the  amount  used  in  the  test  is  so  small  that  it  will  not 
interfere  with  the  reading  of  the  reaction. 

Impregnating  the  Filter  Paper. — The  filter  paper  used 
for  this  purpose  is  Schleich  and  Schull's  No.  597,  but  any 
other  make  of  paper  of  approximately  the  same  texture 
will  serve  equally  as  well.  The  paper  is  cut  into  squares 
measuring  10  by  10  centimeters,  and  actual  experience  has 
shown  that  it  mil  take  about  1.5  c.c.  of  the  rabbit  serum 
to  saturate  one  of  these  squares  of  paper,  so  that  it  is  an 
easy  matter  to  calculate  how  many  squares  will  be  required 
for  any  given  amount  of  the  serum.  This  number  is  placed, 
one  by  one,  in  a  suitable  Petrie  dish  or  other  glass  dish 
containing  the  serum,  and  the  process  continued  until  all 
the  serum  is  absorbed  by  the  paper.  The  squares  of  sat- 
urated paper  are  then  carefully  lifted  with  a  pair  of  for- 
ceps, thoroughly  drained,  and  drawn  across  the  edge  of 
the  dish  a  few  times  to  remove  any  excess  of  serum,  and 
then    placed    upon  a  piece  of  unbleached  muslin,  which 


Fig.    5. 
Same  centrifuge  ilKistrated  in   Fig.   4,  with  shaker  head  attached,  for  use  in  making 

antigenic  extracts. 


PREPAEATIOF   AND   TITRATION    OF   REAGENTS  71 

should  be  large  enough  to  contain  all  the  slips  of  paper. 
The  muslin  containing  the  paper  is  now  placed  under  an 
electric  fan  and  the  paper  rapidly  dried,  after  which  it 
should  be  placed  in  air-tight  glass  containers  and  kept  at 
room  temperature  in  a  dark  and  dry  place.  Before  use, 
it  should  be  titrated  as  will  be  described. 

The  amboceptor  paper  thus  prepared  will  retain  its 
working  strength  for  many  months,  but  it  should  be  ti- 
trated at  least  once  a  month  in  order  to  avoid  any  sudden 
loss  of  strength,  although  the  writer  has  never  observed 
such  losses  in  the  many  years  that  he  has  used  the  ambo- 
ceptor paper.  Paper  over  a  year  old  has  been  repeatedly 
used  and  in  practically  the  same  dose  as  when  it  was  first 
prepared  and  paper  two  years  old  was  found  to  have  lost 
only  one-eighth  of  its  original  strength  at  the  end  of  that 
time. 

Preparation  of  the  Hemol3rtic  Antigen. — The  hemolytic 
antigen  used  in  the  test  consists  of  a  5  per  cent  suspension 
of  human  red  blood  corpuscles,  prepared  in  the  following 
manner : 

In  the  Wassermann  test,  as  recommended  by  the  writer, 
each  tube  contains  0.1  c.c.  of  a  5  per  cent  suspension  of 
human  red  blood  corpuscles  in  0.85  per  cent  salt  solution. 
The  quantity  of  blood  required  for  a  series  of  tests  will 
depend,  of  course,  upon  their  number.  One  cubic  centi- 
meter of  blood  will  furnish  enough  suspension  for  nearly 
one  hundred  tests,  and  unless  one  is  doing  a  very  large 
number  of  tests,  enough  blood  may  be  easily  secured  by 
constricting  the  middle  finger,  after  forcibly  swinging  the 
arm  about  for  a  few  times,  with  a  rubber  tube,  and  prick- 
ing the  finger  just  above  the  root  of  the  nail  with  a  sterile 
needle  or  glass  point.  The  blood  is  allowed  to  drop  into 
sterile  graduated  centrifuge  tubes,  each  tube  being  filled  to 
the  9  c.c.  mark  with  sterile  normal  salt  solution  (0.85%), 
and  enough  blood  is  added  to  fill  the  tube  to  the  10  c.c.  mark. 
The  tubes  are  then  shaken  well  and  centrifuged  until  all 
the  blood  corpuscles  are  collected  at  the  bottom.    The  su- 


72  THE   WASSERMANN   TEST 

pernatant  liquid  is  then  carefully  poured,  or  pipetted  off, 
the  tubes  again  filled  with  salt  solution,  and  again  centri- 
fuged  until  the  cells  are  at  the  bottom  of  the  tube.  This 
process  of  washing  is  repeated  until  the  corpuscles  have 
been  washed  four  times,  which  will  be  found  sufficient. 

The  supernatant  fluid  is  poured  or  pipetted  off  for  the 
last  time,  and  the  amount  of  corpuscles  at  the  bottom  of 
the  tube  noted,  and  the  tube  filled  with  enough  salt  solution 
to  make  a  5  per  cent  suspension.  Thus,  if  there  are  0.5  c.c. 
of  red  corpuscles  at  the  bottom  of  the  tube,  it  would  require 
9.5  c.c.  of  salt  solution  to  make  a  5  per  cent  suspension. 
If  only  a  small  amount  of  blood  is  needed  for  a  few  tests, 
one  centrifuge  tube  will  be  all  that  is  required  for  the 
washing  but  if  many  tests  are  to  be  performed,  the  wash- 
ing should  be  done  with  a  greater  number  of  tubes. 

In  the  test,  0.1  c.c.  of  this  suspension  is  added  to  each 
tube,  which  contains  0.9  c.c.  of  normal  salt  solution,  thus 
making  a  half  of  one  per  cent  suspension.  This  makes  a 
suspension  sufficiently  strong  to  render  the  reading  of  the 
test  easy  and  is  much  more  saving  of  complement  and  am- 
boceptor than  the  use  of  a  stronger  suspension. 

Preparation  of  Antigens  for  Wassermann  Test. — The 
syphilis  antigen,  as  it  may  be  called,  as  already  stated,  is 
not  a  true  antigen,  in  the  strict  use  of  the  term,  as  it  is  an 
extract  containing  lipoids  which  react  with  lipotropic  sub- 
stances in  the  blood  serum  of  the  syphilitic  patient,  but  as 
it  acts  like  a  true  antigen  in  the  Wassermann  test,  it  is 
convenient  to  retain  the  name,  but  it  should  be  understood 
that  it  is  not  a  real  antigen,  as  are  the  red  blood  corpuscles 
used  in  the  hemolytic  system  just  described. 

It  is  certainly  true  that  in  all  alcoholic  extracts  of  nor- 
mal tissues  used  as  antigens  in  the  Wassermann  test,  the 
active  principle  is  contained  within  lipoids  in  the  extracts 
and  that  there  is  nothing  specific  as  regards  Treponema 
pallidum,  in  these  extracts.  When  alcoholic  extracts  of 
foetal  syphilitic  liver,  and  especially  watery  extracts  of 
such  livers,  are  used  as  antigens,  the  extracts,  besides  con- 


Fig.  6. 
Small   incubator,  to   run   at   56°    C,   foi    inactivating   sera. 


PKEPARATION   AND   TITRATION    OF   REAGENTS  73 

taining  the  lipoids  present  in  the  normal  extracts,  may  also 
contain  specific  antigenic  substances  due  to  Treponema 
pallidum,  as  has  already  been  sho^^m.  However,  even  in 
such  extracts,  the  greater  part  of  the  reaction  is  "undoubt- 
edly due  to  the  lipoids  present  in  them,  although  it  can  not 
be  denied  that  in  some  instances,  where  the  extracts  of 
normal  tissue  give  a  negative  or  doubtful  result,  the  ex- 
tracts of  foetal  sj'-philitic  liver  and  of  cultures  of  Trepo- 
nema pallidum  give  a  positive  one.  Accordingly,  the 
writer  advises  the  nse  of  both  a  cholesterinized  alcoholic 
extract  of  normal  human  heart  muscle  and  an  alcoholic 
extract  of  foetal  syphilitic  liver  in  i)erforming  the  test.  If 
both  can  not  be  obtained  the  best  results  will  be  secured 
by  using  cholesterinized  alcoholic  extracts  of  normal  tis- 
sues, preferably  human  heart  muscle.  The  extracts  of  pure 
cultures  of  Treponema  pallidum,  while  giving  interesting 
results  from  a  scientific  standpoint,  are  useless  as  antigen 
in  routine  Wassermann  work  because  of  their  unreliability. 
In  practical  Avork  the  writer  recommends  the  antigens  al- 
ready mentioned,  and,  in  addition,  Noguchi's  acetone- 
insoluble  lipoid  antigen,  where  the  other  antigens  fail  in 
apparently  true  syphilitic  infections,  but  only  in  such  in- 
fections. 

Alcoholic  Extract  of  Foetal  Syphilitic  Liver. — A  foetal 
liver,  rich  in  Treponema  pallidum  is  selected,  washed  free 
from  blood,  and  any  fat  present  removed.  One  hundred 
grams  is  weighed  out,  all  blood  removed  b^^  washing,  and 
cut  into  very  small  pieces.  The  material  is  then  placed  in 
a  suitable  bottle,  or  other  container,  and  1000  grams  of 
absolute  alcohol,  or  95%  alcohol,  added,  and  the  whole 
placed  in  an  incubator  at  37°  C.  and  allowed  to  remain  for 
10  days,  being  thoroughly  shaken  three  times  a  day  during 
that  time,  when  it  will  be  ready  to  titrate.  If  a  shaking 
machine  is  available,  the  material,  after  adding  the  alco- 
hol, is  placed  in  a  suitable  bottle  or  bottles,  and  shaken  for 
24  hours  at  a  good  rate  of  speed,  at  the  end  of  which  time 
it  will  be  found  that  the  tissue  is  thoroughly  extracted. 


74  THE   WASSERMANN   TEST 

After  extraction  "by  either  method  is  completed,  the  mate- 
rial is  filtered  through  filter  paper  and  the  filtrate  titrated. 
If  the  first  titration  is  not  satisfactory,  the  filtrate  should 
be  evaporated  to  two-thirds  its  original  volume  and  again 
titrated.  The  antigenic  extract  should  always  he  kept  in 
the  ice  box  except  when  in  use. 

Cholesterinized  Human  Heart  Extract. — The  same 
method  is  used  in  preparing  the  alcoholic  extract  of  heart 
muscle,  the  material  being  washed  free  from  blood,  all  fat 
removed,  and  100  grams  of  the  heart  muscle  being  cut  into 
small  pieces  and  extracted  with  alcohol  in  the  same  pro- 
portions (1000  grams  of  alcohol  to  100  grams  of  muscle). 
After  extraction  one-half  of  the  material  is  filtered  through 
paper,  and  0.4%  of  cholesterin  added,  and  titrated.  If  the 
titration  does  not  result  satisfactorily  as  regards  the  anti- 
genic strength  of  the  extract,  the  remaining  half  is  evapo- 
rated to  two-thirds  its  original  volume,  again  filtered 
through  paper,  and  the  cholesterin  added,  after  which  it 
is  titrated.  The  amount  of  cholesterin  added  gives  prac- 
tically a  saturated  solution  and  there  is  generally  an  ex- 
cess of  cholesterin  left  after  standing,  and  in  using  the 
antigen  care  should  be  taken  not  to  get  any  of  this  excess 
into  the  pipette  used  in  the  work  and  thus  in  the  diluted 
solution  of  the  antigen.  After  the  addition  of  the  choles- 
terin the  antigenic  extract  is  placed  in  glass-stoppered  bot- 
tles and  kept  in  an  ice  box,  except  when  in  use. 

Noguchi's  Acetone  Insoluble  Lipoid  Antigen. — This  type 
of  antigen  was  recommended  by  Noguchi  when  he  evolved 
his  modification  of  the  AVassermann  test  and  it  is  an  excel- 
lent antigen,  especially  for  use  in  cases  in  which  the 
ordinary  antigens  described  give  doubtful  results,  as  it 
appears  to  be  more  delicate  than  the  extracts  already  de- 
scribed. However,  it  is  true  that  sometimes  this  antigen 
appears  to  give  positive  reactions  in  cases  that  are  nega- 
tive with  other  antigens  and  in  which  syphilis  can  not  be 
stated  to  exist  upon  clinical  grounds,  so  that,  in  the  writ- 
er's opinion,  the  use  of  this  antigen  should  be  restricted  to 


Fig.  7. 
Water-bath,   suitable   for   Wassermann   test   incubations. 


PREPARATION   AND   TITRATION   OF   REAGENTS  75 

the  class  of  cases  mentioned  and  as  a  control  of  other  anti- 
gens, when  doubtful  results  are  obtained.  It  is  prepared 
in  the  following  manner: 

Normal  organs  are  used  and  ma^^  be  from  man  or  the 
lower  animals.  Extracts  made  from  heart  muscle,  liver, 
or  kidney  of  man  or  the  same  organs  of  the  guinea  pig  or 
rabbit  are  satisfactory  and  the  extraction  is  done  in  the 
same  manner  as  for  the  antigens  already  described,  10 
parts  of  absolute  alcohol  being  used  for  each  part  of  tis- 
sue. After  extraction,  the  material  is  filtered,  and  the 
filtrate  evaporated  to  drjmess  by  the  aid  of  an  electric  fan. 
The  residue  is  taken  up  with  ether  and  the  solution  thus 
obtained  is  allowed  to  stand  over  night  in  a  cool  place.  In 
the  morning  the  supernatant  ether  will  be  found  perfectly 
clear  while  the  insoluble  portion  of  the  residue  has  settled 
to  the  bottom  of  the  receptacle.  The  clear  etheral  portion 
is  decanted  off  and  evaporated  to  a  small  quantity  and 
mixed  with  ten  volumes  of  pure  acetone.  The  precipitate 
which  forms  is  allowed  to  settle  and  the  supernatant  liquid 
poured  off.  The  precipitate  is  light  brown  in  color  and 
forms  the  material  from  which  the  antigen  suspension  is 
made,  0.3  gram  of  this  acetone  insoluble  fraction  being  dis- 
solved in  1  c.c.  of  ether  and  then  mixed  with  9  c.c.  of  methyl 
alcohol.  This  mixture  may  be  kept  for  a  long  time  and 
the  antigen  suspension  used  in  making  the  test  is  prepared 
by  adding  1  c.c.  of  this  stock  solution  to  9  c.c.  of  normal 
salt  solution,  or  if  less  is  needed  at  the  time  of  making  the 
tests,  the  same  proportion  of  antigen  and  salt  is  preserved. 

Wassermann's  Aqueous  Extract  Antigen. — The  antigen 
employed  by  Wassermann  in  his  method  of  performing  the 
complement-fixation  test  for  syphilis  was  prepared  as 
follows : 

A  foetal  syphilitic  liver,  rich  in  treponemas,  is  cut  into 
very  small  pieces  and  weighed.  Four  times  its  weight  of 
0.5  per  cent  phenol  in  physiologic  salt  solution  is  added 
and  the  mixture  placed  in  a  brown  bottle  and  shaken  in  a 
shaking   machine   at   room   temperature   for   twenty-four 


76  THE   WASSERMANN   TEST 

hours.  It  is  then  filtered  through  ganze  and  placed  in  a 
brown  bottle  in  the  ice  box  for  several  days,  after  which  it 
is  titrated  to  determine  its  antigenic  strength.  A  sediment 
forms  in  the  bottle  after  standing  in  the  ice  box  and  one 
shonld  be  careful  not  to  disturb  this  in  using  the  antigen. 

This  antigen  is  not  satisfactory,  as  it  varies  very  greatly 
in  strength  when  made  from  different  livers,  and  is  very 
prone  to  lose  strength  rapidty,  especially  if  it  is  left  for 
any  length  of  time  at  room  temperature.  It  is  not  nearly 
as  satisfactory  as  the  antigens  heretofore  described. 

Various  other  methods  of  preparing  antigenic  extracts 
for  the  Wassermann  test  have  been  described  by  Marie 
and  Levaditi,*^  Landsteiner,  Miiller,  and  Potzl,**^  Porges 
and  Meier,^^  Bro^vning,  Cruickshank,  and  McKenzie,*^  and 
others,  but  none  of  them  are  better  in  practical  use  than 
those  which  have  been  treated  of  in  the  foregoing  para- 
graphs. 

Cholesterinized  Antigens. — Before  concluding  this  por- 
tion of  our  subject  it  may  be  well  to  discuss  briefly  the  sub- 
ject of  the  use  of  cholesterinized  antigens  in  the  Wasser- 
mann test.  The  writer  advocates  their  use,  or,  at  least,  the 
use  of  an  extract  of  human  heart  muscle,  cholesterinized,  be- 
cause he  has  found  by  long  experience  that  such  an  extract 
is  perfectly  reliable  and  does  not  give  false  positive  reac- 
tions, as  shown  by  the  results  in  the  many  thousands  of 
tests  in  which  he  has  used  such  an  extract.  Some  author- 
ities claim  that,  in  their  experience,  cholesterinized  ex- 
tracts are  so  very  sensitive  that  they  may  give  false  posi- 
tive results  but  this  has  not  been  the  ^vriter's  experience. 
Very  rarely  there  has  been  observed  a  plus  or  plus-minus 
reaction  in  apparently  normal  serum,  but  the  writer  has 
never  observed  a  distinctly  positive  reaction  Avith  the 
cholesterinized  heart  extracts  used  in  his  method  in  a  se- 
rum which  could  be  proved  to  be  normal,  but,  on  the  other 
hand,  numerous  instances  have  been  observed  in  which 
ordinary  extracts  gave  doubtful  or  negative  results  but  in 
which  the  cholesterinized  heart  extract  gave  positive  re- 


PREPARATION   AND   TITRATION   OF   REAGENTS  77 

actions.  This  is  very  apt  to  be  the  case  in  cases  of  syphilis 
that  have  been  treated  and,  in  the  writer's  opinion,  the 
cholesterinized  extract  is  far  superior,  as  a  control  of 
treatment,  than  are  extracts  of  normal  tissues  or  of  foetal 
syphilitic  liver,  to  which  cholesterin  has  not  been  added. 

Titration  of  Reagents  Used  in  the  Wassermann  Test. 

The  accurate  titration  of  the  various  reagents  used  in 
the  Wassermann  test  is  of  fundamental  importance,  as  the 
test  is  essentially  a  quantitative  one  and  the  smallest  vari- 
ation in  the  amount  of  the  reagents  used  may  cause  very 
great  variations  in  the  result  of  the  test.  If  the  amount 
of  complement  or  amboceptor  be  excessive  the  strongest 
positive  serum  may  give  a  negative  result,  while  if  the 
amount  of  these  reagents  is  too  small,  normal  sera  will 
give  positive  results.  The  same  is  true  of  the  amount  of 
antigen,  for  an  excess  of  antigen  will  result  in  a  positive 
result  in  normal  sera  or  in  the  blood  sera  from  other  dis- 
eases than  syphilis,  while  too  little  will  result  in  negative 
reactions  in  what  should  be  found  to  be  strongly  positive 
sera. 

It  is  easily  seen,  therefore,  that  the  accurate  titration  of 
complement,  amboceptor,  and  antigen  is  of  the  greatest 
practical  importance,  and  the  neglect  of  such  titrations  will 
be  sure  to  lead  to  erroneous  results  and  to  great  injustice 
being  done  the  patient  who  has  entrusted  the  testing  of  his 
blood  to  the  laboratory. 

Titration  of  the  Complement. — The  amount  of  comple- 
ment to  be  used  in  the  Wassermann  test  is  of  vital  impor- 
tance and  is  determined  by  a  titration  of  the  guinea  pig 
serum  each  day  before  the  tests  are  made.  Some  author- 
ities employ  exactly  one  unit  of  complement  with  one  unit 
of  amboceptor  for  each  test  but  this  alloAvs  of  no  inhibitory 
effect  by  either  the  blood  serum  or  antigen,  and,  is  there- 
fore, a  dangerous  proceeding  unless  very  carefully  con- 
trolled. If  the  titrations  are  made  with  the  amount  of 
patient's  serum  added  to  the  complement  and  amboceptor 
the   fallacy   residing   in   the   possible   anticomplementary 


PLATE  I 

Fig.  1. — Titration  of  Complement.  Each  of  the  tubes  contains  the  amount 
of  complement  marked  above  the  tube.  In  the  titration,  as  illustrated,  hemo- 
lysis is  complete  in  the  fourth  tube,  containing  0.05  c.e.  of  complement.  Hence 
0.05  c.e.  is  the  complement  unit  and  twice  this  amount  is  used  in  the  actual 
test,  or  0.1  c.e. 

The  tenth  tube  is  a  control  tube  containing  no  complement  and  it  should 
show  complete  inhibition  of  hemolysis,  as  illustrated. 

Fig.  2. — Titration  of  Amboceptor  Papers.  Each  of  the  first  five  tubes  con- 
tains a  piece  of  amboceptor  paper  of  the  size  indicated  above  the  tubes.  In  the 
titration  illustrated  the  first  tube  showing  complete  hemolysis  is  the  third  tube, 
containing  a  piece  of  paper  measuring  5  by  3  mm.  Hence  the  unit  of  ambo- 
ceptor paper  is  a  piece  measuring  5  by  3  nun.,  and  twice  this  amount  is  used 
in  the  test,  or  a  piece  measuring  5  by  6  mm. 

The  sixth  tube  is  a  control  tube  containing  no  amboceptor  paper  and  should 
show  complete  inhibition  of  hemolysis,  as  illustrated. 

Fig.  3. — 'Titration  of  Antigen.  Tubes  1,  2,  3,  and  4  contain  respectively 
0.05,  0.1,  0.15,  and  0.2  c.e,  of  a  1  in  10  dilution  of  the  antigenic  extract,  with 
0.1  c.e.  of  a  known  syphilitic  serum.  Tube  5  contains  0.1  c.e.  of  the  syphilitic 
serum  without  antigen.  Tube  6  contains  0.1  c.e.  of  a  normal  blood  serum  with 
0.2  c.e.  of  the  diluted  antigen.  Tube  7  contains  0.1  c.e.  of  the  normal  serum 
without  antigen.  Tube  8  contains  complement  without  amboceptor,  while 
Tube  9  contains  the  hemolytic  system,  acting  as  a  control  of  the  latter, 

A  good  antigen  should  give  the  results  illustrated  in  this  titration,  causing 
complete  inhibition  of  hemolysis  in  the  presence  of  0.05  c.e.  of  a  syphilitic 
serum,  as  shown  in  Tube  1,  and  no  inhibition  of  hemolysis  with  a  normal  serum 
in  amounts  as  large  as  0.2  c.e,  as  shown  in  Tube  6.  Complete  inhibition  of 
hemolysis  should  occur  in  Tube  8  and  complete  hemolysis  in  5,  6,  7,  and  9. 


'  P»^"  "H  w 


«p» 


0-oZcc.o-o3cc.  0-0^c.c.0'05cc.  o-o6cx:.007c.c   ooda:  oo9c.c   o/cc    Control 

n 


*«  -„^'j^ 


v^  v_y  w 

Fig. I.  Tltrai-ion    of     Complement. 

5x1mm.     5xZmm.    5x3mn7.    SxH-mm.    5x5mm.    Control 

n 


pi  '»»m'       '■mnjmmmia         yp 


9  ^-"M^ 


^  r— Ijsi 


^w^     v^     m/ 

Fig.E.  Titrafion  of  Amboceptor  Papers. 
1         z         3         ¥ 


%■«    "-^  ' 


Sp/  s^  w^  w^  ^w  ^V  ^iv  W/ 

Fig. 3.   Titration    of  Antigen. 


Plate  I. 


PREPAKATION   AND   TITRATION   OF   REAGENTS  79 

action  of  the  blood  serum  would  be  overcome,  but  this  is 
a  cumbersome  procedure,  especially  when  a  large  number 
of  tests  are  to  be  made,  and,  in  addition,  there  is  still  the 
factor  of  the  natural  inhibition  of  hemolysis  which  is  pres- 
ent in  almost  all  antigens.  It  is,  therefore,  much  better 
to  use  an  excess  of  complement  in  making  the  tests  and  it 
has  been  found  perfectly  safe  in  practice  to  double  the 
unit  of  complement  found  by  titration,  when  actually  do- 
ing the  test.  In  other  words,  two  units  of  complement  are 
used  for  each  tube  employed  in  the  test. 

In  order  to  titrate  the  complement  it  is  necessary  that  a 
standard  amount  of  amboceptor  and  a  standard  suspension 
of  human  red  blood  corpuscles  be  adopted  and  strictly  ad- 
hered to,  although  the  strength  of  the  blood  suspension 
may  be  varied,  provided  the  complement  be  titrated  with 
it  before  each  series  of  tests.  Standard  doses  of  ambo- 
ceptor are  adopted  because  this  reagent  changes  little  in 
strength  during  long  periods  of  time,  while  if  the  ambo- 
ceptor were  to  be  titrated  before  each  series  of  tests,  a 
standard  dose  of  complement  would  have  to  be  adopted 
and  the  latter  varies  so  greatly  in  strength  that  it  is  pref- 
erable to  titrate  the  complement  mth  a  standard  dose  of 
amboceptor. 

In  the  titration  of  the  complement  two  units  of  ambo- 
ceptor, as  determined  by  titration,  are  employed,  or  the 
amount  of  amboceptor  that  is  actually  used  in  the  test. 
The  complement,  amboceptor,  and  blood  cells  should  be  all 
added  at  once,  for  if  the  amboceptor  and  the  blood  cells 
are  left  in  contact,  without  the  complement,  for  any  length 
of  time,  the  cells  will  becom.e  "sensitized"  and  a  smaller 
amount  of  complement  will  produce  hemolysis. 

In  making  the  titration  of  the  complement,  either  a  5  or 
10  per  cent  suspension  of  human  erythrocytes,  in  normal 
salt  solution,  may  be  used,  but  the  writer  prefers  the  5  per 
cent  suspension  as  it  is  more  economic  of  amboceptor  and 
is  of  sufficient  strength  to  render  the  readings  of  the  reac- 
tion easy.    It  should  be  remembered  that  the  stronger  the 


80 


THE   WASSERMANN"   TEST 


blood  suspension  is  the  greater  will  be  the  amount  of  am- 
boceptor and  complement  necessary  to  produce  hemolysis, 
and  the  more  extravagant  the  expenditure  of  these  re- 
agents, so  that  it  is  foolish  to  employ  a  stronger  blood  sus- 
pension for  the  test  than  is  necessary  to  enable  one  to 
readily  read  the  reactions,  as  the  hemolytic  system  is 
merely  used  as  an  indicator  of  the  presence  in  the  patient's 
blood  of  the  substances  that  absorb  or  bind  complement. 
In  titrating  the  complement,  test  tubes  measuring  100 
by  12.5  millimeters  are  used.  These  tubes  should  be  per- 
fectly clean  and,  while  they  do  not  need  to  be  sterile,  the 
best  technic  is  to  have  them  sterilized.  The  complement 
is  diluted  with  an  equal  amount  of  normal  salt  solution 
(0.85%)  and  graduated  amounts  are  added  to  tubes  con- 
taining the  blood  suspension  and  amboceptor,  as  illustrated 
in  Table  XVI. 

Table  XVI 
Titration  of  the  Complement 


AMOUNT    OF 

AMOUNT    OF 

NUMBER    OP 

AMOUNT    OF 

TUBE 

SALT     SOLU- 

BLOOD    SUS- 

AMBOCEPTOR 

COMPLEMENT 

Incubate     i  n 

NO. 

TION 

PENSION,  5% 

UNITS 

C.C. 

water-bath      a  t 

CO. 

c.c. 

37°   C.  for  one 

1 

0.9 

0.1 

2 

0.02 

hour   and   read. 

2 

0.9 

0.1 

2 

0.03 

The  tube  show- 

3 

0.9 

0.1 

2 

0.04 

i  n  g      complete 

4 

0.9 

0.1 

2 

0.05 

hemolysis     con- 

5 

0.9 

0.1 

2 

0.06 

tains    one    unit 

6 

0.9 

0.1 

2 

0.07 

of    complement. 

7 

0.9 

0.1 

2 

0.08 

Two    units    are 

8 

0.9 

0.1 

2 

0.09 

used  in  making 

9 

0.9 

0.1 

2 

0.10 

the  test. 

10 

0.9 

0.1 

0 

0.10 

After  incubating  in  the  water-bath  at  37°  C.  for  one 
hour,  or  in  the  ordinary  bacteriological  incubator,  for  two 
hours  at  the  same  temperature,  the  titration  is  read,  and 
the  tube  that  contains  the  smallest  amount  of  complement 
which  shows  complete  hemolysis  is  noted  and  this  amount 
is  called  one  unit  of  complement.  For  instance,  if  it  is 
found  that  Tube  No.  4,  containing  0.05  c.c.  of  complement, 


Fig.   S. 
Amboceptor    cutter.      Front    view    showing   gauge.      Devised    by    Lieut.    Colonel    E.    B. 
Vedder,  Medical  Corps,  U.   S.  Army,  for  cutting  amboceptor  paper.      (From  Bulletin  No    8, 
War  Department,   Washington,   D.    C,   Office   of  the   Surgeon   General,   U.    S.   Army,   1915  ) 


PREPARATION   AND   TITRATION   OF   REAGENTS  81 

is  the  first  of  the  tubes  to  show  complete  hemolysis,  the 
tubes  preceding  it  showing  only  partial  hemolysis,  then 
0.05  c.c.  of  complement  constitutes  one  unit  of  complement, 
and  in  performing  the  actual  test  twice  this  amount  or 
0.1  c.c.  will  be  used  for  the  complement  dose  for  each  tube, 
or  two  units  of  complement.  The  amount  of  complement 
is  doubled  in  order  to  provide  for  the  anticomplementary 
action  of  the  patient's  blood  serum  and  of  the  antigen,  and 
has  been  found  in  practice  to  be  sufficient  to  answer  this 
purpose  .  and  at  the  same  time  not  to  cause  fallacies  in 
the  test. 

In  reading  the  titration  if  there  is  the  slightest  cloud- 
iness in  any  of  the  tubes  hemolysis  can  not  be  said  to  be 
complete  and  in  selecting  the  tube  which  represents  one 
complement  unit,  the  first  that  shows  an  absolutely  crystal 
clear  solution  should  be  noted.  The  control  tube.  Tube  No. 
10,  which  contains  no  amboceptor,  should  show  complete 
inhibition  of  hemolysis.  If  it  does  not,  there  is  something 
wrong  with  the  complement  or  the  salt  solution  used  in 
making  the  titration,  and  the  titration  should  be  repeated 
with  new  reagents. 

This  Titration  of  the  Complement  Must  be  Made  Before 
Each  Series  of  Tests  or  Very  Serious  Errors  Will  De- 
velop.— The  blood  serum  of  guinea  pigs  varies  greatly  in 
complementary  strength,  so  that  it  is  excessively  danger- 
ous to  use  a  stated  amount  of  the  serum  for  complement. 
A  common  observation,  upon  titrating  different  samples  of 
guinea  pig  serum,  is  to  find  differences  in  complementary 
strength  all  the  way  from  a  unit  equalling  0.03  c.c.  to  one 
of  0.08  c.c.  and  sometimes  a  serum  will  be  found  much 
stronger  or  weaker  than  these  limits,  so  that  the  danger 
of  using  any  definite  amount  is  apparent.  The  complement 
must  he  carefully  titrated  before  each  series  of  tests. 

Titration  of  the  Hemolytic  Amboceptor. — As  already 
stated,  the  hemolytic  serum  or  amboceptor  used  in  the 
Wassermann  test  as  recommended  by  the  writer  is  pre- 
pared by  immunizing  rabbits  to  human  red  blood  corpus- 


82 


THE   WASSERMANIsr   TEST 


cles.  The  human  hemolytic  system,  as  it  is  called,  was 
adopted  in  order  to  obviate  the  danger  arising  from  the 
natural  antisheep  amboceptor  present  in  human  blood  se- 
rum, and  which  is  frequently  present  in  quantities  suffi- 
cient to  render  a  positive  syphilitic  serum  negative  unless 
special  precautions  are  taken  to  guard  against  such  an 
occurrence.  While  this  result  is  not  frequent  in  practice, 
when  the  sheep  hemol^^tic  system  is  used,  the  fact  that  it 
may  occur,  and  that,  in  order  to  be  perfectly  safe,  it  is 
necessary  to  remove  from  each  patient's  blood  the  anti- 
sheep  amboceptor  that  may  be  present,  renders  the  use 
of  the  human  hemolytic  system  preferable,  both  from  a 
scientific  and  practical  point  of  view,  and  it  is  the  system 
the  writer  has  always  urged  for  complement-fixation  tests 
upon  human  blood  serum. 

The  hemolytic  amboceptor,  having  been  prepared  by 
impregnating  suitable  filter  paper  with  it,  as  already  de- 
scribed, must  be  titrated  in  order  to  determine  the  ambo- 
ceptor unit.  After  the  paper  is  thoroughly  dry  a  strip  is 
cut  from  one  of  the  sheets  measuring  5  millimeters  in 
width  and  varying  lengths  of  this  strip  are  used  in  the 
titration,  as  shoA\Ti  in  Table  XVII.  The  outer  edge  of  the 
saturated  sheet  of  filter  paper  should  be  trimmed  off  be- 
fore cutting  the  strip  referred  to,  in  order  to  avoid  any 
concentration  of  the  serum  which  might  occur  at  that  por- 
tion of  the  paper. 

Table  XVII 
Titration  of  Amboceptor  Paper* 


TUBK 
NO. 


amount  of 
salt   solu- 
TION 
CO. 


0.9 
0.9 
0.9 
0.9 
0.9 
0.9 


AMOUNT    OF 

COMPLEMENT 

UNITS 


AMOUNT    OF 
BLOOD     SUS- 
PENSION 
C.C. 


0.1 
0.1 
0.1 
0.1 
0.1 
0.1 


AMOUNT    OF 

AMBOCEPTOR 

PAPER 

MM. 


by  1 
by  2 
by  3 
by  4 
by  5 
None 


Incubate  i  n 
water-bath  a  t 
37°  C.  for  one 
hour  or  in  incu- 
bator for  two 
hours  at  same 
temp  erature, 
and  read  re- . 
suits. 


*If  the  unit  of  complement  has  not  been  determined  against  a  known  amboceptor  by 
titration,  use  0.05  c.c.  of  a  1:1  dilution  of  the  complement  as  the  unit.  It  may  be  neces- 
sary to  carry  the  titration  on  further  by  using  larger  amounts  of  the  amboceptor  paper, 
but  a  good  paper  should  give  hemolysis  in  a  piece  5  by  5  millimeters. 


Fig,   9. 
Amboceptor    cutter.      Rear    view,    showing    scale    and    set    screw.      Devised    by    Lieut. 
Colonel    e;.    B.    Vedder,    Medical    Corps,    U.    S.    Arrny.      (From    Bulletin    No.    8,    War    De- 
partment,  Office  of  the   Surgeon  General,  U.   S.  Army,  Washington,   D.   C.) 


PREPARATIOIT   AND   TITRATIOl^r   OF   REAGE:N'TS  83 

After  incubating  in  the  water-bath  at  37°  C.  for  one 
hour,  or  in  the  incubator,  at  the  same  temperature,  for  two 
hours,  the  tubes  being  shaken  every  fifteen  minutes  in  or- 
der to  facilitate  the  liberation  of  the  amboceptor  serum, 
the  titration  is  read  and  the  first  tube  to  show  complete 
hemolysis  is  noted  and  the  amount  of  the  paper  contained 
in  that  tube  is  called  one  amboceptor  unit.  A  good  ambo- 
ceptor paper  should  show  complete  hemolysis  in  tubes 
Nos.  2  or  3,  or  with  pieces  of  paper  measuring  5  by  2  or 
3  millimeters,  but  paper  measuring  5  by  5  millimeters  can 
be  used  in  the  test.  In  many  instances,  the  first  tube  will 
show  complete  hemolysis  and,  when  this  occurs,  the  paper 
may  be  reduced  in  width,  or  the  strength  of  the  blood  sus- 
pension may  be  increased,  a  ten  per  cent  suspension  being 
used  instead  of  a  five  per  cent  suspension.  The  control 
tube,  No.  6,  should  not  show  any  hemolysis  and  if  it  does 
it  indicates  that  something  is  wrong  with  the  blood  sus- 
pension or  the  complement,  and  the  titration  mil  have  to 
be  repeated  mth  new  reagents. 

In  making  the  actual  Wassermann  test  tivo  units  of 
amboceptor  paper  should  be  used  in  order  to  allow  for  the 
anticomplementary  qualities  of  human  blood  serum  and  of 
the  antigen.  Thus,  if  one  unit  of  the  paper  was  found  to 
be  a  piece  measuring  5  by  2  mm.  a  piece  5  b}^  4  mm.  should 
be  used  in  making  the  test. 

Amboceptor  paper,  being  very  stable,  does  not  need  to 
be  titrated  very  frequently,  once  a  month  being  amply  suf- 
ficient, and  such  paper  will  keep,  with  little  loss  of  strength, 
for  several  months. 

Titration  of  the  Syphilitic  Antigen. — The  antigens  used 
in  the  Wassermann  test  must  be  carefully  titrated  and 
such  titrations  should  be  made  at  frequent  intervals,  as  all 
antigens  used  in  the  test  lose  strength  with  more  or  less 
rapidity.  To  be  on  the  safe  side,  the  antigen  should  be 
titrated  at  least  once  in  two  weeks  unless  it  be  found  that 
the  particular  antigens  that  are  employed  are  very  stable, 
when  monthly  titrations  may  be  sufficient.    A  really  good 


84 


THE   WASSERMANN   TEST 


antigen  loses  strength  slowly  and  the  writer  has  often 
worked  with  extracts  that  were  practically  as  strong  in 
antigenic  qualities  after  several  months  as  when  first  pre- 
pared, but  the  reverse  is  often  true,  and  an  antigen  will 
be  found  that  rapidly  loses  strength,  thus  making  frequent 
titrations  advisable.  If  possible,  it  is  best  to  titrate  the 
antigen  before  each  series  of  tests. 

An  antigen  must  be  titrated  with  a  known  syphilitic  se- 
rum in  order  to  determine  its  antigenic  qualities;  with  a 
normal  serum  in  order  to  demonstrate  that  it  will  not  in- 
hibit hemolysis  when  used  with  such  a  serum;  and  in  the 
absence  of  either  a  syphilitic  or  normal  serum,  in  order  to 
determine  whether  or  not  it  is  anticomplementary  or  hemo- 
lytic of  itself.  Before  titrating,  the  antigen  should  be 
properly  diluted,  which  is  accomplished  by  adding  one  part 
of  the  extract  to  nine  parts  of  normal  salt  solution  (0.85%). 
Any  antigenic  extract  prepared  in  the  manner  recom- 
mended by  the  Avriter  and  diluted  so  as  to  produce  an 
emulsion  containing  one  part  of  the  extract  to  nine  parts 
of  salt  solution,  that  is  neither  anticomplementary  nor 
hemolytic  of  itself  or  in  the  presence  of  a  normal  serum 
in  a  dose  of  0.2  c.c.  and  that  gives  inhibition  of  hemolysis 
in  the  presence  of  a  syphilitic  serum  in  a  dose  of  0.05  or 
0.1  c.c,  will  be  found  satisfactory  in  practice. 


Table  xvni 
Titration  op  Antigen  for  Hemolytic  Properties 


AMOUNT    OF 

AMOUNT    OF 

AMOUNT    OF 

TUBE 

SALT     SOLU- 

UNITS OF 

ANTIGEN 

BLOOD     SUS- 

Incubate    i  n 

NO. 

TION 

COMPLEMENT 

EMULSION 

PENSION.  5% 

water-bath     a  t 

C.C. 

CO. 

C.C. 

37°   C.  for  one 

1 

0.9 

2 

0.05 

0.1 

hour    or   in  in- 

2 

0.9 

2 

0.10 

0.1 

cubator  for  two 

3 

0.9 

2 

0.15 

0.1 

hours,  and  read 

4 

0.9 

2 

0.20 

0.1 

results. 

5 

0.9 

2 

None 

0.1 

Table  XVIII  illustrates  the  titration  of  antigenic  ex- 
tracts for  the  purpose  of  determining  their  hemolytic 
properties.     The    quantities    of    antigenic  solution  tested 


Fig.   10. 
Amboceptor  markers.     Devised  by  Lieut.   Colonel   E.    B.   \'edder,   Medical   Corps,   U.    S. 
Army,   for  accurately  measuring  units   of  amboceptor  jiaper.      (From   Bulletin  No.    8,   War 
Department,   Office   of  the   Surgeon   General,   U.   S.   Army,    Washington,    D.    C.) 


PREPARATION   AND   TITRATION   OF   REAGENTS 


85 


have  been  determined  by  a  large  number  of  experimental 
tests  to  yield  satisfactory  results. 

In  this  titration  none  of  the  tubes  should  show  hemolysis. 
If  hemolysis  occurs  in  all,  including  Tube  5,  the  control 
tube  without  antigen,  it  would  demonstrate  that  hemolytic 
substances  were  present  either  in  the  complement  or  blood 
suspension,  or  both ;  but  if  hemolysis  should  occur  in  one 
or  more  of  the  tubes  containing  the  antigen,  the  control 
tube  remaining  unhemolyzed,  it  would  demonstrate  that 
the  antigenic  extract  is  hemolytic;  and,  in  this  event,  it 
should  be  discarded  unless,  upon  being  further  diluted  with 
salt  solution,  it  is  found  to  give  satisfactory  results.  If 
none  of  the  tubes  containing  antigen  show  hemolysis,  the 
antigen  is  not  hemolytic,  and  therefore,  so  far  as  hemo- 
lytic properties  are  concerned,  it  is  safe  to  use  in  making 
the  Wassermann  test. 

In  order  to  determine  whether  antigens  are  anticomple- 
mentary or  not,  they  are  titrated  as  shown  in  Table  XIX. 


Table  XIX 
Titration  of  Antigen  for  Anticomplementary  Properties 


TUBE 

no. 


amount  of 
salt  so- 
lution 
c.c. 

UNITS    OF 
COMPLE- 
MENT 

amount  of 
antigenic 
emulsion 

C.C. 

AMOUNT  OF 
BLOOD    SUS- 
PENSION, 

5% 

C.C. 

UNITS  OF 
AMBOCEP- 
TOR PAPER 

0.9 

2 

0.05 

0.1 

2 

0.9 

2 

0.10 

0.1 

2 

0.9 

2 

0.15 

0.1 

2 

0.9 

2 

0.20 

0.1 

2 

0.9 

2 

None 

0.1 

2 

Incub  a  1 13 
1  n  water- 
bath  at  37° 
C.  for  one 
hour  or  in 
incu  b  a  t  0  r 
for  two 
hours  at 
same  t  e  m- 
perature  and 
read   results. 


As  the  result  of  this  titration  there  should  be  complete 
hemolysis  in  all  of  the  tubes.  If  Tube  5,  the  control  tube, 
should  show  inhibition  of  hemolysis,  it  would  prove  that 
either  the  complement,  amboceptor,  or  blood  suspension, 
was  inhibitory,  but  if  any  of  the  tubes  containing  antigen 
show  inhibition,  the  control  tube  being  hemolyzed,  it  would 
demonstrate  that  the  antigen  is  anticomplementary,  and, 
unless  further  dilution  will  remove  this  property  without 


86  THE   WASSERMANN   TEST 

injuring  its  antigenic  value,  the  extract  should  not  be  used 
for  the  test. 

Having  tested  an  antigen  for  its  hemolytic  and  anticom- 
plementary properties,  it  is  now  necessary  to  titrate  it 
against  a  known  syphilitic  blood  serum  to  determine  its 
antigenic  value,  and  with  a  known  normal  blood  serum  to 
demonstrate  that  it  will  not  give  a  positive  reaction 
with  nonsyphilitic  sera.  This  titration  is  illustrated  in 
Table  XX. 

As  the  result  of  this  titration  all  of  the  tubes  containing 
antigen  should  show  complete  inhibition  of  hemolysis  in 
which  syphilitic  blood  serum  is  also  present  and  complete 
hemolysis  in  the  tubes  in  which  normal  blood  serum  is 
present.  Thus  Tubes  1,  2,  3,  and  4  should  show  absolute 
inhibition  of  hemolysis,  while  Tube  6  should  show  complete 
hemolysis.  If  Tube  6  shows  any  trace  of  inhibition,  the 
antigen  is  not  suitable  and  should  be  discarded;  and  the 
same  is  true  if  there  is  less  than  complete  inhibition  of 
hemolysis  in  Tube  2.  There  may  be  a  slight  trace  of  hem- 
olysis in  Tube  1  containing  only  0.05  c.c.  of  the  antigenic 
emulsion,  without  harm ;  but  it  mil  always  be  found  better 
in  practice  to  use  only  those  antigenic  extracts  that  give 
complete  inhibition  of  hemolysis  with  0.05  of  the  diluted 
antigen.  Tube  7,  which  is  a  control  of  the  normal  serum 
used,  should  show  complete  hemolysis,  and  if  it  does  not, 
another  serum  should  be  tested.  Tube  8,  the  control  of  the 
complement,  should  show  complete  inhibition  of  hemolysis ; 
and  Tube  9,  the  control  of  the  hemolytic  system,  should 
show  complete  hemolysis. 

If  these  results  are  obtained,  they  demonstrate  that  the 
antigen,  as  diluted,  is  suitable  for  use  in  the  Wassermann 
test  as  recommended  by  the  writer,  in  that  it  produces 
inhibition  of  hemolysis  in  the  presence  of  syphilitic  blood 
serum  in  a  dose  as  small  as  0.05  c.c,  and  that  it  does  not 
inhibit  hemolysis  in  the  presence  of  a  normal  serum  in  four 
times  that  dose,  or  0.2  c.c.  Such  an  antigen  will  give  excel- 
lent results  and  even  one  that  gives  inhibition  of  hemolysis 


PREPAKATION   AND   TITRATIOlSr   OF   REAGENTS 


87 


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00  THE   WASSERMANN   TEST 

with  a  syphilitic  blood  serum  in  a  dose  of  0.1  will  be  found 
satisfactory  in  practice,  provided  it  is  otherwise  all  right. 

The  amount  of  antigenic  emulsion  necessary  to  produce 
complete  inhibition  of  hemolysis  in  the  presence  of  a  syph- 
ilitic blood  serum  is  known  as  an  antigen  unit.  The  anti- 
gen unit  is  not  doubled  in  mahing  the  test  as  is\  the  unit 
of  complement  and  amboceptor. 

All  blood  sera  used  in  titrating  the  antigen  should  be 
inactivated  by  heating  for  one-half  hour  at  56°  C.  and  a 
neglect  of  this  will  produce  most  erroneous  results. 

Antigens  should  be  kept  in  the  ice  box  when  not  in  use 
as  exposure  to  room  temperature  for  any  length  of  time 
will  result  in  their  rapidly  losing  strength. 


CHAPTER  V 

THE  TECHNIC  OF  THE  WRITER'S  MODIFICATION 
OF  THE  WASSERMANN  TEST.     THE   TEST 
UPON    THE    CEREBROSPINAL    FLUID. 
THE     ORIGINAL     WASSERMANN 
TECHNIC.     OTHER  MODIFI- 
CATIONS OF  THE  WAS- 
SERMANN TEST. 

The  technic  of  the  writer's  modification  of  the  Wasser- 
maim  test  was  first  fully  published  in  1913,  in  Bulletin 
No.  3,  War  Department,  Office  of  the  Surgeon  General, 
entitled  Studies  in  Syphilis,  by  Charles  F.  Craig,  Captain, 
Medical  Corps,  U.  S.  Army  and  Henry  J.  Nichols,  Cap^ 
tain.  Medical  Corps,  U.  S.  A.  The  method  has  been  used 
by  the  writer  since  1910  wUh  success  and  since  1913  it  has 
been  the  standard  method  adopted  in  nearly  all  of  the  large 
army  laboratories,  and  has  been  recommended  for  use  in 
all  army  laboratories  by  Lieut.  Col.  Vedder,  of  the  Medical 
Corps,  who  has  devised  special  apparatus  for  use  with  the 
method.  It  is  a  modification  of  both  the  original  Wasser- 
mann  method  and  of  that  of  Noguchi,  following  Wasser- 
mann  in  using  an  extract  of  foetal  syphilitic  liver  as  one 
antigen  and  in  inactivating  the  patient's  blood  serum,  and 
Noguchi  in  using  a  human  hemolytic  system  instead  of  the 
sheep  system. 

This  modification  of  the  Wassermann  test  has  been  so 
thoroughly  tried  in  the  army,  and  so  many  thousands  of 
tests  have  been  made  with  it,  that  there  is  no  question  of 
its  accuracy  and  value  in  the  diagnosis  of  syphilis.  Many 
different  laboratory  workers  have  used  it  and  their  re- 
sults have  all  been  in  agreement  as  to  its  accuracy  and 


90  THE   WASSERMANl^   TEST 

as,  in  army  practice,  we  make  many  repeated  examina- 
tions of  the  same  case  in  order  to  control  the  treatment, 
any  irregularities  in  the  results  of  the  test  would  have  been 
soon  discovered.  In  the  hands  of  different  workers  the  re- 
sults have  been  almost  identical  with  the  same  character  of 
cases,  as  is  instanced  by  Vedder,*^  and  it  is  believed  that  it 
is  as  well  adapted  to  civilian  laboratories  as  to  those  of  the 
army  and  will  be  found  at  once  simple  and  accurate. 

The  method  of  preparing  the  reagents  used  in  the  test 
has  already  been  described,  as  well  as  their  titration,  and 
in  this  chapter  will  be  considered  the  exact  technic  of  the 
test  using  the  reagents  as  prepared  and  titrated. 

The  glassware  used  in  preparing  the  various  reagents 
for  the  test  and  in  the  test  should  be  perfectly  clean.  No 
chemicals  should  be  used  in  cleaning  the  glassware  but 
only  hot  water  and  soap,  followed  by  a  thorough  rinsing 
with  hot  water.  All  glassware,  except  the  test  tubes  used 
in  the  test,  should  be  sterilized  by  dry  heat  and  one  should 
be  careful  that  a  temperature  of  180°  C.  is  not  exceeded, 
as  a  higher  temperature  than  this  is  apt  to  so  heat  the 
ends  of  the  small  1  c.  c.  pipettes  as  to  close  them.  The 
test  tubes  do  not  need  to  be  sterilized,  and  after  washing, 
should  not  be  plugged,  but  placed  open  end  downward  in 
a  test  tube  basket  and  covered  with  a  clean  towel  when  not 
in  use. 

The  pipettes  used  for  measuring  the  patient's  serum 
should  be  graduated  in  tenths  and  do  not  need  to  be  grad- 
uated in  hundredths,  thus  saving  considerable  expense 
where  many  tests  are  being  done,  as  an  individual  pipette 
should  be  used  with  each  patient's  blood  serum.  At  least 
a  dozen  1  c.c.  pipettes,  graduated  plainly  in  hundredths, 
the  graduations  being  at  such  a  distance  apart  that  0.02 
c.c.  can  be  accurately  measured,  should  be  in  stock,  for 
titration  work  and  in  making  dilutions.  In  using  any 
pipette  graduated  to  the  tip  the  last  unit  of  graduation 
should  not  be  used  as  it  is  very  apt  to  be  inaccurate.  Great 
care  should  be  exercised  in  using  the  pipettes  to  avoid 


TECHNIC    OF   THE   WRITER'S   MODIFICATION  91 

breakage,  especially  of  the  tip,  and  they  should  not  be  al- 
lowed to  remain  unwashed  after  using  but  should  be  im- 
mediately cleaned,  as  otherwise  the  blood  serum  will  dry 
in  them  and  will  be  found  very  difficult  to  remove  owing 
to  the  small  caliber  of  the  pipette. 

Collection  and  Preparation  of  the  Patient's  Blood  Se- 
rum.— When  specimens  of  blood  for  the  Wassermann  test 
are  to  be  sent  through  the  mail  or  kept  for  more  than  two 
days  before  they  are  tested,  the  blood  should  be  collected 
with  aseptic  precautions  and  placed  in  sterile  glass  con- 
tainers. If  the  test  is  to  be  made  at  once  or  within  a  day 
or  two  such  precautions  are  not  necessary  but  it  is  always 
essential  that  an  aseptic  technic  be  employed  in  collecting 
the  blood,  in  order  to  avoid  any  danger  of  infection  to  the 
patient.  The  reason  for  observing  these  precautions  has 
been  noted  in  the  discussion  of  the  effect  of  contaminating 
bacteria  upon  the  result  of  the  Wassermann  test.  All 
specimens  of  blood  serum  for  the  test  should  be  kept  in  the 
ice  box  until  used,  as  exposure  to  room  temperature  may 
cause  the  serum  to  become  anticomplementary. 

Before  taking  the  blood  from  the  patient  he  should  be 
questioned  regarding  the  use  of  alcohol  and  if  he  states 
that  he  has  used  alcoholics  within  thirty-six  hours  the 
blood  should  not  be  taken  but  the  patient  told  to  return 
upon  the  follomng  day,  meanwhile  abstaining  from  the 
use  of  alcohol  in  any  form.  This  precaution  is  necessary 
owing  to  the  fact  that  alcohol  is  capable  of  rendering  a 
positive  reaction  negative,  as  shown  by  the  writer  and 
Nichols  and  already  discussed. 

Patients  undergoing  treatment  for  syphilis  frequently 
give  a  negative  Wassermann  reaction  during  the  time  treat- 
ment is  being  administered,  so  that  it  is  better  to  test  the 
blood  serum  after  treatment  has  been  stopped  for  a  period 
of  two  or  three  weeks.  Blood  for  the  Wassermann  test 
should  not  be  taken  during  a  malarial  paroxysm,  as  some- 
times a  positive  reaction  is  given  in  such  instances,  nor 
should  it  be  collected  immediately  after  anesthesia  or  after 


92  THE   WASSERMANN   TEST 

eating.  Bile  stained  blood  sera  may  inhibit  hemolysis  and 
should  not  be  tested  if  it  is  possible  to  secure  a  specimen 
from  the  patient  that  is  not  bile  stained.  If  a  positive  re- 
action occurs  mth  a  bile  stained  serum  it  should  be  re- 
membered that  it  may  be  a  false  positive  and  the  diagnosis 
of  syphilis  should  only  be  made  provisionally. 

Blood  serum  should  not  be  over  five  or  six  days  old,  at 
the  most,  when  tested,  as  older  sera  frequently  are  anti- 
complementary. If  a  serum  inhibits  hemolysis  in  both  the 
antigen  and  control  tube  another  specimen  should  be  re- 
quested. Inactivation  often  removes  the  anticomplemen- 
tary properties  of  old  sera  but  it  is  best  not  to  employ 
them  in  the  test  if  it  is  possible  to  avoid  doing  so. 

Collection  of  the  Blood. — The  amount  of  blood  serum 
required  in  the  writer's  modification  of  the  Wassermann 
test  does  not  exceed  0.2  c.c.  so  that  2  c.c.  of  blood  is  amply 
sufficient  for  the  test.  This  amount  may  be  easily  collected 
from  a  puncture  wdth  a  needle  or  glass  needle  just  above 
the  finger  nail  upon  the  dorsal  surface  of  the  middle  finger 
in  the  adult,  allomng  the  blood  to  drop  into  a  small,  sterile 
and  perfectly  dry  glass  vial  until  from  one  and  one-half  to 
two  cubic  centimeters  are  collected,  or  the  blood  may  be 
collected  from  a  puncture  in  the  lobe  of  the  ear  with  a 
Wright  tube.  Any  form  of  special  apparatus  that  has  been 
devised  for  collecting  blood  for  the  Wassermann  test  is 
unnecessary  and  if  venipuncture  is  objected  to  by  the  pa- 
tient enough  blood  can  easily  be  obtained  as  described 
above. 

In  the  writer's  opinion,  the  simplest  and  best  method 
of  obtaining  blood  for  the  test  is  to  remove  from  2  to  5 
c.c.  of  blood  from  one  of  the  large  veins  in  the  forearm  or 
at  the  bend  of  the  elbow  with  a  sterilized  glass  syringe. 
The  arm  should  be  cleaned,  the  site  of  the  puncture  of  the 
vein  brushed  with  iodine,  and  the  needle,  held  almost  par- 
allel with  the  surface  of  the  arm,  pushed  through  the  skin 
and  directly  into  the  vein.  The  blood  should  be  withdrawn 
slowly  or  the  syringe  can  be  dispensed  with  entirely  and 


TECHNIC   OF   THE   WEITER's   MODIFICATION"  93 

the  blood  allowed  to  flow  directly  into  a  container  from 
the  needle.  The  operation  is  practically  painless  and  with 
a  little  practice  anyone  can  become  very  expert  in  the  pro- 
cedure. The  advantage  of  the  method  is  that  more  blood 
can  be  collected,  thus  insuring  an  abundant  supply  of  the 
serum  to  be  tested. 

After  collection,  the  blood  is  placed  in  a  suitable  glass 
vial,  the  serum  allowed  to  separate,  after  which  it  is 
pipetted  into  a  sterile  glass  vial  or  tube  if  it  is  to  be  mailed, 
or  used  at  once  in  the  test.  The  serum  separates  best  if 
the  blood  be  kept  at  room  temperature  for  an  hour  and 
then  placed  in  the  ice  box.  All  blood  serum  should  be  kept 
in  the  ice  box  until  used,  and  when  specimens  are  received 
by  mail,  they  should  be  immediately  placed  in  the  ice  box. 
A  positive  and  negative  blood  serum  should  ahvays  be 
kept  in  stock  for  control  sera  and  these  should  be  kept  in 
the  ice  box  when  not  in  use.  All  blood  sera  used  for  the 
Wassermann  test  should  be  clear  in  appearance  and  with 
no  disagreeable  odor.  Milky  or  cloudy  sera  are  often  due 
to  chyle  but  it  is  best,  if  possible,  not  to  use  such  sera, 
although  they  may  not  cause  inhibition  of  hemolysis. 

Inactivation  of  the  Blood  Serum. — All  blood  sera  used 
in  the  Wassermann  test  as  recommended  by  the  luriter 
must  be  inactivated  by  heating  at  56°  C.  for  one-half  hour. 
This  is  done  for  the  purpose  of  removing  the  native  com- 
plement in  the  blood  serum  and  also  for  removing  any 
thermolabile  anticomplementary  substances  that  may  have 
developed  through  age  or  bacterial  contamination.  While 
the  inactivation  of  the  serum  also  tends  to  make  it  weaker 
in  substances  that  give  a  positive  reaction  mth  syphilitic 
serum,  as  so  well  shown  by  Noguchi,^°  it  is  absolutely  nec- 
essary that  inactivation  be  employed  in  any  modification 
of  the  Wassermann  test  that  employs  either  aqueous  or 
alcoholic  extracts  of  tissues  ivhich  contain  proteins  and 
this  is  true  of  the  original  Wassermami  test  and  most  of 
its  modifications.  When  Noguchi's  acetone  insoluble  anti- 
genic extract  is  used  the  blood  serum  does  not  require  in- 


94  THE   WASSERMANN"   TEST 

activation  and  this  is  one  of  the  advantages  that  its  author 
claims  for  it.  However,  in  practice,  the  amount  of  harm 
done  by  inactivation,  in  weakening  the  reacting  power  of 
the  blood  serum,  is  small  when  compared  with  the  harm 
which  would  result  in  using  active  serum,  and  it  is  only 
in  very  early  infections  or  in  those  which  have  received 
treatment  that  inactivation  might  so  weaken  the  serum  as 
to  render  it  negative. 

In  inactivating  the  blood  serum  extreme  care  should  be 
taken  that  56°  C.  is  not  exceeded,  for  exposure  of  the 
serum  to  a  temperature  of  60°  C.  will  practically  destroy 
its  power  of  reacting  w^ith  syphilitic  serum.  The  temper- 
ature may  be  allowed  to  stand  at  55°  C.  with  as  good 
results  as  at  56°  C,  but  the  latter  figure  should  never  be 
exceeded. 

At  the  same  time  that  the  sera  to  be  tested  are  inac- 
tivated, a  knoAvn  syphilitic  serum  and  a  known  normal 
serum  are  also  inactivated,  to  serve  as  controls  of  the 
test.  In  addition,  there  is  always  pro\dded  a  tube  for 
the  serum  to  be  tested,  in  which  no  antigen  is  placed,  the 
control  tube,  and  this  is  chiefl}^  for  the  purpose  of  deter- 
mining that  the  serum  tested  is  not  anticomplementary  of 
itself.  This  control  tube  of  each  serum  tested  should  never 
be  omitted  for  if  it  is  no  dependence  can  be  placed  upon 
the  results  of  the  test. 

The  Writer's  Technic. — Having  inactivated  the  sera 
to  be  tested  and  the  positive  and  normal  control,  and  being 
provided  with  the  proper  blood  suspension,  hemolytic  am- 
boceptor, and  syphilitic  antigens,  the  test  can  be  proceeded 
with. 

Before  doing  anything  else,  hoivever,  the  complement 
must  be  titrated  as  already  described  and  the  exact  comple- 
ment unit  determined,  and  this  is  doubled  for  each  tube  in 
making  the  test.  This  procedure  should  never  be  omitted 
under  any  consideration.  Some  authorities  prefer  to  ti- 
trate the  amboceptor  instead  of  the  complement,  but  as  the 


TECHFIC    OF   THE   WRITER'S   MODIFICATION  95 

complement  is  the  substance  in  the  test  that  is  most  apt  to 
vary  in  strength,  it  should  be  titrated  in  preference  to  the 
amboceptor.  As  a  matter  of  fact,  the  titration  of  the  com- 
plement with  a  fixed  dose  of  amboceptor,  also  determined 
by  previous  titration,  can  not  result  in  any  fallacies  in  the 
test,  as  the  amount  of  complement  necessary  to  produce 
hemolysis  with  the  dose  of  amboceptor  used,  when  doubled, 
is  always  sufficient  to  give  accurate  results  when  used  in 
the  test. 

It  is  recommended  that  two  antigens  be  used  in  testing 
each  serum,  one,  the  alcoholic  extract  of  foetal  syphilitic 
liver;  the  other,  the  alcoholic  extract  of  normal  human 
heart  muscle,  to  which  0.4  per  cent  of  cholesterin  has  been 
added.  If  both  antigens  can  not  be  obtained,  good  prac- 
tical results  can  be  obtained  by  using  one,  as  it  is  seldom 
that  a  positive  result  is  obtained  ^\4th  one  antigen  and  not 
with  the  other. 

If  two  antigens  are  used  three  test  tubes  are  needed  for 
each  serum  to  be  tested,  while  if  only  one  antigen  is  used, 
two  tubes  will  be  sufficient.  The  test  tubes  should  be  placed 
in  racks  having  a  double  row,  an  anterior  and  a  posterior. 
When  three  tubes  are  used,  two  should  be  placed  in  the  an- 
terior row  of  the  rack  and  one  in  the  posterior,  while  if 
two  tubes  are  used,  one  should  be  placed  anteriorly  and 
one  posteriorly.  The  anterior  tubes  contain  the  patient's 
blood  serum  together  mth  the  syphilitic  antigen, .  while  the 
posterior  contains  the  patient's  blood  serum  without  the 
antigen,  this  tube  acting  as  a  control  of  the  serum  tested. 
Besides  the  tubes  for  the  patient's  blood  serum,  there 
must  be  an  equal  number  of  tubes  for  a  kno^vn  syphilitic 
serum,  the  positive  control ;  and  for  a  known  normal  serum, 
the  normal  serum  control. 

In  making  the  test  proceed  as  follows :  In  all  of  the 
tubes  enumerated  place  0.9  c.c.  of  normal  salt  solution 
(0.85%).  In  Tube  1  and  Tube  2,  anterior,  if  two  antigens 
are  used,  or  in  Tube  1,  if  only  one  antigen  is  used,  place 
0.1  c.c.  of  the  inactivated  blood  serum  of  the  patient  to  be 


96  THE   WASSEKMANN   TEST 

tested,  and  the  same  amount  in  Tube  1,  posterior.  The 
same  amount  of  syphilitic  and  normal  blood  serum  is 
placed  in  the  control  tubes  mentioned  and  in  the  same  man- 
ner. Thus,  if  two  antigens  are  used,  in  Tubes  3  and  4 
0.1  c.c  of  the  known  syphilitic  serum  is  placed,  and  the 
same  amount  in  Tube  3,  posterior,  while  if  only  one  anti- 
gen is  used,  0.1  c.c.  of  the  syphilitic  serum  is  placed  in 
Tube  2,  anterior,  and  in  Tube  2,  posterior.  There  is  now 
added  to  every  tube  used  in  the  test  tivo  units  of  comple- 
ment and  to  each  anterior  tube  one  unit  of  the  antigen  used. 
If  two  antigens  are  used,  one  unit  of  each  should  be  placed 
in  the  anterior  tubes,  alternately. 

The  tubes  are  now  placed  in  a  water-bath  at  37°  C.  for 
one  half  hour,  or  in  an  ordinary  bacteriological  incubator, 
at  the  same  temperature,  for  one  hour,  and  at  the  expira- 
tion of  this  time  there  is  added  to  each  tube  0.1  c.c.  of  the 
5  per  cent  suspension  of  human  red  blood  corpuscles  and 
two  units  of  the  amboceptor  paper.  The  tubes  are  again  in- 
cubated in  the  water-bath  at  37°  C,  this  time  for  one  hour, 
or  in  the  incubator  for  two  hours,  being  shaken  every  fif- 
teen minutes  in  order  to  facilitate  the  liberation  of  the 
amboceptor  serum  from  the  filter  paper.  After  this  incu- 
bation the  tubes  are  placed  in  an  ice  box  for  one  or  two 
hours  and  the  results  then  read.  Neglect  of  shaking  the 
tubes,  as  recommended,  will  oftentimes  result  in  partial 
'positive  reactions  in  sera  that  should  react  negatively. 

If  desired  a  hemolytic  and  antigen  control  tube  may  be 
included  but  this  is  not  necessary  in  practice  as  the  titra- 
tions of  the  complement  will  show^  if  anything  is  wrong 
with  the  hemolytic  system,  and  an  antigen  control  is  unnec- 
essary unless  it  has  been  some  time  since  the  antigen  was 
titrated. 

Results  of  the  Test. — If  all  of  the  reagents  used  in  the 
test  are  working  properly  the  results,  after  the  time  al- 
lowed in  the  ice  box,  should  be  as  follows : 

The  anterior  tube  or  tubes,  containing  the  patient 's  blood 


TEST    UPON    THE    CEREBROSPINAL   FLUID  97 

serum,  and  the  antigen  or  antigens,  should  show  complete 
inhibition  of  hemolysis  (positive  reaction),  if  syphilis  is 
present;  while  the  posterior  tube,  containing  the  patient's 
serum  without  antigen,  should  show  complete  hemolysis. 
Of  course,  various  degrees  of  inhibition  will  be  shown  in 
the  anterior  tube  or  tubes  if  the  case  is  an  early  one,  treat- 
ment has  been  given,  or  if  the  reaction  is  weak.  If  the 
patient  is  not  syphilitic  there  should  be  complete  hemolysis 
in  the  anterior  tube  or  tubes  as  well  as  in  the  posterior 
tube.  The  posterior,  or  control  tube  of  the  patient's  se- 
rum, should  always  show  complete  hemolysis,  and  if  it  does 
not,  another  specimen  should  be  requested. 

The  anterior  tube,  or  tubes,  containing  the  kno^m  syph- 
ilitic blood  serum,  or  the  positive  control,  should  show 
complete  inhibition  of  hemolysis  (positive  reaction)  while 
the  posterior  tube  of  the  same  serum  mthout  antigen, 
should  show  complete  hemolysis.  If  inhibition  occurs  in 
this  tube  it  demonstrates  that  the  control  positive  serum  is 
worthless. 

The  anterior  tube,  or  tubes,  containing  the  kno\\m  nor- 
mal serum,  or  the  normal  control,  with  antigen,  should 
show  complete  hemolysis,  as  should  the  posterior  tube  of 
this  set. 

If  a  control  tube  for  the  antigen  and  for  the  hemolytic 
system  be  used,  both  should  show  complete  hemolysis. 

The  Test  Upon  the  Cerebrospinal  Fluid 

The  great  value  of  the  Wassermann  test  when  per- 
formed with  the  cerebrospinal  fluid  in  demonstrating  the' 
existence  of  syphilitic  infection  of  the  central  nervous  sys- 
tem renders  it  almost  imperative  that,  before  assuring  a 
patient  that  he  is  free  from  infection,  the  spinal  fluid  be 
tested.  Not  only  is  this  true,  but  the  fluid  should  be  tested 
in  all  cases  early  in  the  infection,  if  possible,  in  order  to 
ascertain  whether  the  nervous  system  is  involved,  and  if 
this  were  more  generally  done,  it  is  probable  that  many  of 


PLATE  II  N 

Fig.  1. — Eeading  of  Results  of  Wassermann  Test.  The  first  tube  shows  com- 
plete inhibition  of  hemolysis  and  may  be  read  four-plus  or  double-plus,  accord- 
ing to  the  nomenclature  used  in  the  individual  laboratory. 

Tubes  2  and  3  show  three-plus  and  two-plus  reactions,  or  both  are  included 
and  reported  as  a  plus  reaction. 

Tubes  4  and  5  show  single-plus  and  plus-minus  reactions,  or  may  both  be 
included  and  reported  as  a  plus-minus  reaction. 

Tube  6  shows  a  minus  or  negative  reaction. 

Fig.  2. — Wassermann  Test.  Positive  Reaction.  Tube  1  contains  the  pa- 
tient 's  blood'  serum  and  syphilitic  antigen  and  shows  complete  inhibition  of 
hemolysis  or  a  positive  reaction.  Tube  2  is  a  control  of  the  patient's  serum 
without  antigen,  and  shows  complete  hemolysis.  Tube  3  is  a  known  syphilitic 
control  serum.  Tube  4  is  a  control  of  the  positive  serum,  without  antigen. 
Tube  5  is  a  known  normal  serum  and  Tube  6  a  control  of  the  normal  serum 
without  antigen. 

Fig.  3. — Wassermann  Test.  Negative  Reaction.  Tube  1  contains  the  pa- 
tient's blood  serum  and  syphilitic  antigen  and  shows  complete  hemolysis,  or  a 
negative  reaction.  Tube  2  is  a  control  of  this  serum  without  antigen.  Tube  3 
is  a  known  positive  serum  and  shows  complete  inhibition  of  hemolysis.  Tube  4 
is  a  control  of  the  known  positive  serum  without  antigen.  Tube  5  is  a  known 
normal  serum  with  the  antigen  and  Tube  6  a  control  of  the  normal  serum 
without  antigen. 


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Fig.l   Reading  of  Results  of  Wassermann  Tesf 

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Fig.3  Wassermann  Test  Negative  Reaction 

Plate  II. 


TEST   UPOIST   THE    CEREBROSPINAL   FLUID  99 

the  severe  complications  characteristic  of  involvement  of 
the  central  nervous  system  could  he  avoided. 

Collection  of  the  Cerebrospinal  Fluid. — The  cerebrospinal 
fluid  is  collected  by  lumbar  puncture  and  when  properly 
performed,  this  operation  is  devoid  of  danger.  The  pa- 
tient should  be  in  bed  and  should  remain  in  bed  for  twenty- 
four  hours  after  the  puncture.  The  writer  is  well  aware  of 
the  fact  that  scores  of  punctures  of  the  spinal  canal  are 
made  in  the  physician's  office,  the  patient  afterward  being 
allowed  to  walk  home  or  to  attend  to  his  business,  but,  in 
his  opinion,  this  practice  is  unjustifiable  and  should  be  dis- 
couraged, for  while  the  danger  of  complications  may  be 
small,  deaths  have  occurred  during  or  shortly  after  the 
procedure  when  thus  performed,  and  severe  symptoms  are 
not  uncommon. 

The  lumbar  region  is  washed  carefully  with  soap  and 
water,  alcohol  and  ether,  and  the  region  over  the  lumbar 
vertebrae  brushed  with  iodine  solution.  The  site  of  punc- 
ture is  most  easily  ascertained  by  running  the  finger  along 
the  spines  of  the  vertebrae  until  the  so-called  "soft  spot" 
is  reached,  which  is  indicated  by  a  distinct  feeling  of  soft- 
ness to  the  finger  and  is  situated  between  the  third  and 
fourth  lumbar  vertebrae.  The  patient  may  sit  upon  a 
stool  with  his  back  bent  so  as  to  bring  the  spinous  proc- 
esses of  the  vertebrae  close  to  the  surface,  or  lie  upon  the 
left  side  upon  the  edge  of  the  bed.  The  latter  posture  is 
necessary  in  the  case  of  the  sick  but  the  sitting  posture 
is  suitable  for  ordinary  ambulant  patients.  The  needle 
used  in  the  puncture  should  be  of  flexible  steel  measuring 
10  cm.  long,  with  a  bore  of  1  to  1.5  mm.,  while  for  a  child 
the  needle  should  be  shorter  but  of  the  same  bore.  The 
needle  should  be  sterilized  before  being  used,  as  well  as 
the  stylet  which  should  accompany  it. 

The  space  between  the  third  and  fourth  lumbar  vertebra 
having  been  ascertained  the  puncture  is  made  directly  in 
the  median  line,  which  I  have  found  much  more  satisfac- 
tory than  the  lateral  puncture  recommended  by  many  writ- 


100  THE   WASSEEMANIiJ"   TEST 

ers.  The  needle  is  held  firmly  and  the  puncture  made 
quickly,  the  direction  being  straight  forward.  Enough 
force  should  be  used  to  push  the  needle  quickly  through  the 
skin  and  muscles  but  as  soon  as  the  spinal  ligaments  are 
reached  it  may  be  pushed  forward  more  slowly  until  there 
is  a  sudden  sensation  of  loss  of  resistance  when  the  needle 
is  in  the  spinal  canal.  The  stylet  is  now  withdrawn  and 
the  spinal  fluid,  if  the  puncture  is  successful,  will  flow  from 
the  needle.  If  it  does  not  flow,  the  needle  may  be  shifted 
very  gently  or  the  patient  told  to  take  a  deep  breath,  or  the 
stylet  may  be  replaced  in  the  needle  and  gently  pushed 
forward  to  clear  the  tube  of  any  materials  that  have  been 
carried  into  it  by  the  influx  of  the  spinal  fluid.  If  none  of 
these  procedures  are  successful  the  puncture  is  said  to  be 
a  *'dry"  one  and  there  is  little  use  in  repeating  it. 

Not  over  5  c.c.  of  fluid  should  be  withdra"v^m,  as  that  is 
amply  sufficient  for  the  test  and  the  less  fluid  withdrawn 
the  slighter  will  be  the  symptoms  following  the  puncture. 
The  fluid  often  comes  from  the  needle  under  considerable 
pressure  and  this  should  be  noted  upon  the  slip  sent  to  the 
laboratory  with  the  fluid.  It  should  be  free  from  blood  but 
can  be  used  even  if  blood  corpuscles  are  present,  by  centri- 
fuging  the  fluid  until  they  are  deposited  at  the  bottom  of 
the  tube  and  pipetting  off  the  clear  fluid. 

The  spinal  fluid  is  kept  in  the  ice  box  until  used  and 
should  not  be  inactivated,  as  is  the  blood  serum,  before  it 
is  tested. 

The  same  procedure  is  followed  in  testing  spinal  fluid 
as  in  testing  blood  serum,  but  different  quantities  of  the 
fluid  are  tested,  as  in  some  infections  of  the  central  nerv- 
ous system,  as  paresis,  a  very  small  amount  of  the  fluid  will 
give  a  positive  reaction,  while  the  same  amount  in  cere- 
brospinal syphilis  would  be  negative.  Therefore  it  has 
always  been  the  writer's  practice  to  test  0.2,  0.5,  0.7  and 
1  c.c.  of  the  spinal  fluid,  thus  making  four  tubes  for  the 
antigen  tubes  and  four  for  the  control  amounts  of  fluid, 


TEST   UPON   THE    CEREBKOSPINAL   ELUID  101 

if  only  one  antigen  is  used,  and  double  this  number  if  two 
antigens  are  used  in  the  test. 

Precautions  to  be  Observed  in  Lumbar  Puncture. — Pa- 
tients who  are  very  weak  or  debilitated  should  not  have  a 
lumbar  puncture  performed  unless  it  is  absolutely  neces- 
sary. Those  suffering  from  paresis  and  tabes  bear  punc- 
ture exceedingly  well  and  seldom  complain  of  pain,  but 
nervous  normal  adults  frequently  speak  of  the  pain  of 
the  puncture  and  of  disagreeable  symptoms  following  it. 
Headache  may  be  severe  if  the  fluid  is  withdrawn  rapidly 
and  if  the  bone  is  punctured  or  bruised  by  the  needle  dur- 
ing the  procedure  pain  may  last  for  several  hours  after- 
wards. Nausea  is  sometimes  observed,  especially  if  the 
fluid  is  rapidly  withdra^^m,  so  that  it  is  always  best  to  allow 
the  fluid  to  flow  as  slowly  as  possible,  thus  relieving 
pressure  gradually.  Nonne^^  warns  against  lumbar  punc- 
ture in  cases  of  cerebral  tumor,  several  deaths  having  fol- 
lowed the  operation  in  this  condition,  and  states  that  he 
has  himself  observed  four  cases  of  sudden  death  in  such 
patients  following  the  puncture. 

The  patient  should  remain  in  bed  for  twenty-four  hours 
after  the  puncture,  if  possible,  and  neglect  of  this  precau- 
tion often  leads  to  disagreeable  s^Tnptoms.  In  a  case  which 
was  observed  in  the  writer's  practice,  very  severe  head- 
aches and  attacks  of  dizziness  occurred  for  several  days 
after  puncture  because  the  patient  insisted  upon  immedi- 
ately returning  to  work.  Diarrhea  is  a  symptom  some- 
times observed,  especially  if  the  fluid  has  been  withdrawn 
rapidly. 

The  Reading  of  the  Reaction  and  Nomenclature  Em- 
ployed.— The  results  of  the  Wassermann  test  are  variously 
reported  from  different  laboratories  and  unless  one  is  ac- 
quainted with  the  meaning  of  the  various  terms  used, 
grave  errors  may  result  in  the  interpretation  of  the  results 
of  the  test. 

In  perhaps  the  large  majority  of  Wassermann  labora- 


102  THE   WASSERMANISr   TEST 

tories  the  results  of  the  test  are  recorded  as  four-plus 
(+  +  +  +),  indicating  complete  inhibition  of  hemolysis,  and, 
therefore,  a  strongly  positive  reaction;  three-plus  (+  +  +), 
indicating  about  75  per  cent  inhibition  of  hemolysis,  or  a 
moderately  positive  reaction;  two-plus  (++),  indicating  50 
per  cent  inhibition  of  hemolysis,  or  a  weakly  positive  re- 
action; plus  (+),  indicating  from  25  to  50  per  cent  inhibition 
of  hemolysis,  or  a  doubtful  reaction;  plus-minus  (+-),  in- 
dicating less  than  25  per  cent  inhibition  of  hemolysis  or  a 
doubtful  reaction;  and  minus  (-),  indicating  complete  hem- 
olysis, or  a  negative  reaction. 

In  the  army  laboratories  a  more  simple  nomenclature 
has  been  employed  for  the  reason  that  only  absolute  inhi- 
bition of  hemolysis  is  considered  a  positive  reaction,  all 
other  degrees  of  inhibition  being  considered  doubtful. 
This  standard  has  been  adopted  principally  that  the  soldier 
may  be  given  the  benefit  of  any  doubt  that  may  exist  re- 
garding syphilitic  infection.  Thus  four  designations  are 
used  in  the  army  laboratories,  as  follows:  Double-plus 
(++),  indicating  complete  inhibition  of  hemolysis,  and, 
therefore,  a  positive  reaction;  plus  (+),  indicating  anything 
between  complete  inhibition  and  50  per  cent  of  hemolysis; 
plus-minus  (+-),  indicating  anything  between  50  per  cent 
of  hemolysis  and  complete  hemolysis;  minus  (-),  indicat- 
ing complete  hemolysis,  or  a  negative  reaction. 

Comparing  this  nomenclature  with  the  foregoing,  the 
three-plus  and  two-plus  reactions  would  correspond  to  our 
plus  reaction,  the  one-plus  with  our  plus-minus  reaction, 
and  the  four-plus  with  our  double-plus  reaction. 

The  writer  believes  that  for  use  in  civilian  life  the  no- 
menclature of  results  first  mentioned,  i.  e.,  four-plus,  three- 
plus,  two-plus,  plus,  plus-minus  and  negative,  is  the  most 
useful;  but  anything  below  a  three- plus  reaction,  in  his, 
opinion,  should  be  considered  doubtful,  unless  there  is  very  ' 
good  evidence  of  syphilitic  infection,  or  the  patient  has 
received  antisyphilitic  treatment. 


ORIGINAL   WASSERMANN   TECHNIC  103 

The  Original  Wassermann  Technic 

In  the  original  Wassermann  technic  an  aqueous  extract 
of  syphilitic  liver  was  employed  as  the  antigen;  the  blood 
sera  were  inactivated  by  heating  them  at  55°  C.  for  one- 
half  hour  before  testing ;  and  a  sheep  hemolytic  system  was 
used.  The  complement  Avas  fresh  guinea  pig  serum  diluted 
1:10  with  normal  salt  solution,  the  dose  being  arbitrarily 
fixed  at  1  c.c.  of  the  diluted  serum.  The  blood  suspension 
was  a  5  per  cent  suspension  of  sheep  erythrocytes  in  nor- 
mal salt  solution,  and  the  dose  for  each  tube  was  1  c.c.  of 
the  suspension.  The  hemolytic  amboceptor  was  the  blood 
serum  of  a  rabbit  immunized  with  sheep's  erythrocytes 
and  this  was  titrated  carefully  before  each  series  of  tests. 
The  antigen,  as  stated,  was  an  aqueous  extract  of  foetal 
syphilitic  liver,  prepared  in  the  manner  already  described 
and  carefully  titrated  before  using.  Two  tubes  were  used 
for  each  of  the  blood  sera  tested ;  two  for  the  positive  con- 
trol ;  two  for  the  normal  control ;  and  three  tubes  for  con- 
trol of  the  hemolytic  system,  antigen  and  blood  suspension. 

The  tubes  are  arranged  in  double  rows  except  the  three 
control  tubes.  For  each  serum  tested  there  should  be  an 
anterior  and  posterior  tube  and  the  same  for  the  known 
positive  serum  and  for  the  known  normal  serum.  In  each 
of  these  tubes  there  is  placed  1  c.c.  of  the  complement,  di- 
luted 1 :10.  In  the  anterior  tube  of  the  test  set  of  two  there 
should  be  placed  0.2  c.c.  of  the  patient's  inactivated  blood 
serum,  and  the  same  amount  in  the  posterior  tube  of  this 
set.  In  the  anterior  tube  and  posterior  tube  of  the  positive 
control  set  there  should  be  placed  0.2  c.c.  of  the  knoAvn  pos- 
itive serum,  and  in  the  two  tubes  of  the  normal  serum  con- 
trol there  should  be  placed  the  same  amount  of  a  known 
negative  blood  serum.  In  the  anterior  tubes  the  proper 
dose  of  antigen  is  placed  and  enough  normal  salt  solution 
to  bring  the  total  amount  in  the  tubes  up  to  3  c.c.  Of  the 
three  control  tubes,  the  first  is  the  antigen  control  and 
should  contain  the  dose  of  antigen  used,  1  c.c.  of  the  com- 


104  THE   WASSERMANN   TEST 

plement,  and  enough  salt  solution  to  equal  3  c.c. ;  the  sec- 
ond is  the  hemolytic  system  control,  and  should  contain 
1  c.c.  of  the  complement  and  2  c.c.  of  the  normal  salt  solu- 
tion; while  the  third  is  the  hlood  suspension  control,  and 
contains  1  c.c.  of  the  corpuscle  suspension  and  2  c.c.  of 
salt  solution. 

The  tubes  are  shaken  and  incubated  for  one  hour  in  the 
incubator  and  to  each  tube  is  then  added  two  units  of  am- 
boceptor serum,  as  determined  by  the  titration  and  1  c.c.  of 
corpuscle  suspension.  The  blood  suspension  control  does 
not,  of  course,  receive  this  suspension.  The  tubes  are 
shaken  and  incubated  until  the  blood  serum  controls  (the 
posterior  tubes)  are  hemolyzed,  and  the  results  read  at 
once  or  after  several  hours  in  the  ice  box. 

If  the  patient's  serum  is  positive,  the  anterior  tube  will 
show  complete  or  almost  complete  inhibition  of  hemolysis, 
while  the  posterior  tube,  containing  the  serum  without  an- 
tigen, should  show  complete  hemolysis;  if  the  patient's 
serum  is  negative,  the  anterior  and  posterior  tubes  should 
both  show  complete  hemolysis.  The  anterior  tube  of  the 
positive  control  set  should  show  complete  inhibition  of 
hemolysis,  while  the  posterior  tube  should  show  complete 
hemolysis;  the  anterior  tube  of  the  normal  serum  control 
and  also  the  posterior  should  show  complete  hemolysis. 
The  antigen  control  tube  should  show  complete  hemolysis, 
as  should  the  hemol^iiic  system  control,  while  the  blood 
suspension  control  should  show  complete  inhibition  of 
hemolysis. 

The  nomenclature  used  in  reporting  the  test  is  that  al- 
ready described,  but  in  reporting  results  to  physicians  it 
is  better  to  report  them  simply  as  positive,  very  suspicious, 
suspicious,  doubtful  or  negative. 

Other  Modifications  of  the  Wassermann  Test 

There  have  been  many  modifications  of  the  original  Was- 
sermann technic  for  the  complement-fixation  test  in  syph- 
ilis and  the  most  important  will  be  here  considered. 


MODIFICATIONS   OF   THE   WASSERMANN   TEST  105 

Nognchi's  Modification. — ^In  Noguchi's  modification  a 
human  hemolytic  system  is  used,  the  serum  to  be  tested  is 
not  inactivated,  and  the  acetone  insoluble  lipoid  antigen  is 
employed,  the  preparation  of  which  has  already  been  de- 
scribed. If  the  blood  serum  can  not  be  tested  at  once,  in- 
activation  should  be  employed  but  better  results  are  ob- 
tained with  active  serum.  Capillary  pipettes  are  used  in 
measuring  the  patient's  serum,  one  drop  of  the  active  se- 
rum being  employed,  or  practically  0.02  c.c.  and  four  drops 
of  the  inactivated  serum,  or  practically  0.08  c.c. 

Noguchi  thus  describes  the  technic  of  the  test:^^  A  rack 
containing  two  rows  of  holes  should  be  used,  an  anterior 
and  a  posterior  row.  ''For  each  test  two  tubes  are  re- 
quired, one  in  the  front  row  and  its  control  in  the  rear 
row.  There  v.dll  also  be  two  pairs  of  tubes  to  serve  as 
positive  and  negative  controls. 

"Put Into  each  of  two  small  test  tubes  front  and  rear  one 
drop  (0.02  c.c.)  of  the  serum  to  be  tested  from  a  capillary 
pipette.  When  using  inactivated  serum  put  4  drops  (0.08 
c.c.)  into  each  tube.  Use  0.2  c.c.  of  cerebrospinal  fluid  not 
inactivated.  Add  to  each  tube  0.1  c.c.  of  40  per  cent  fresh 
guinea  pig  serum  made  by  adding  1  part  of  complement  to 
1^2  parts  of  0.9  per  cent  salt  solution.  To  the  front  tube 
add  the  unit  of  antigen  determined  by  titration.  Then  to 
both  tubes  add  1  c.c.  of  the  one  per  cent  suspension  of 
washed  human  corpuscles  (the  first  procedure).  In  case 
of  using  the  ten  per  cent  corpuscle  suspension  add  0.9  c.c. 
of  salt  solution  and  two  units  of  amboceptor  (the  second 
procedure).  Shake  the  tubes  thoroughly  from  time  to 
time  to  distribute  the  reagents  throughout  the  mixture. 

"With  every  series  of  tests  it  is  necessary  to  carry  two 
sets  of  controls  and  for  this  purpose  four  additional  tubes 
are  necessary.  To  each  of  the  first  pair  of  these,  one  in  the 
front  and  one  in  the  rear  row,  one  capillary  drop  of  a  syph- 
ilitic serum  known  to  give  a  positive  reaction  is  added. 
This  will  serve  as  a  positive  control.    To  the  second  pair 


106  THE   WASSEEMANN"   TEST 

one  drop  of  normal  serum  kno^\Ti  to  give  a  negative  re- 
action should  be  added.  This  tube  will  serve  as  a  negative 
control.  Now  put  into  each  tube  complement  and  into  the 
tubes  of  the  front  row  antigen,  adding  finally  1  c.c.  of  the 
one  per  cent  corpuscle  suspension  (first  procedure),  or  0.9 
c.c.  of  salt  solution  and  2  units  of  amboceptor  (second  pro- 
cedure) to  each  tube.' 

"Place  the  rack  holding  these  pairs  of  tubes  in  a  water- 
bath,  thermostat,  or  warm  place  not  over  37°  C.  Allow  an 
hour  from  the  time  the  mixture  is  made  for  the  antibody  to 
combine  with  the  antigen  and  for  complement  to  be  fixed. 
If  a  water-bath  is  used  30  minutes  is  a  sufficient  length  of 
time.  The  contents  of  the  tubes  in  the  first  procedure 
(first  method)  are  as  follows: 

''Eear.  Test  serum  plus  complement  (2  units)  plus 
1  c.c,  1  per  cent  corpuscle  suspension. 

''Front.  Test  serum  plus  complement  (2  units)  plus 
antigen  plus  1  per  cent  corpuscle  suspension.. 

' '  The  contents  of  the  tubes  in  the  second  procedure  are  as 
follows : 

"Kear.  Test  serum  plus  2  units  complement  plus  2 
units  amboceptor  paper. 

"Front.  Test  serum  plus  complement  (2  units)  plus 
antigen  plus  2  units  amboceptor. 

"First  incubation' at  37°  C.  for  1  hour,  then  add  to  each 
tube  of  the  first  procedure  a  slip  bearing  two  units  of  am- 
boceptor, as  follows : 

"Rear.     Above,  plus  amboceptor  (2  units). 

"Front.     Above,  plus  amboceptor  (2  units). 

' '  To  each  tube  of  the  second  procedure  0.1  c.c.  of  the  ten 
per  cent  corpuscle  suspension,  as  follows : 

"Rear.    Above,  plus  10%  corpuscle  suspension  (.1  c.c). 

"Front.    Above,  plus  10%  corpuscle  suspension  (0.1  c.c). 

"Allow  two  hours  in  the  thermostat  or  one  hour  in  the 
water-bath.  After  final  incubation  the  tubes  are  kept  at 
room  temperature  for  a  few  hours  before  the  results  are 
recorded." 


MODIFICATIOlSrS    OF    THE    WASSERMANN    TEST 


107 


It  will  be  noted  that  Noguclii  gives  two  procedures  or 
methods  of  making  the  test  but  the  first  procedure  is  to 
be  preferred,  in  the  opinion  of  the  writer. 

Table  XII  illustrates  the  method  of  performing  Noguchi's 
luodification  of  the  Wassermann  test  by  the  first  proce- 
dure recommended: 


Table  XXI 

NoGTJCHi  's  Method  of  Performing  the  Wassermann  Test 

Modification 

WITH  His 

set  for  diagnosis 

positive   control 
set 

negative  control 

SET 

o 

o 

o 

d 

CO 

_o 
a 

a 
1— 1 

-*^ 

cS 

O 

3     '^ 

r-      O 

3  -^ 
•"S  o 

o 

hours 

One 

e-half 

Posterior  Bow 

Posterior  Eoiv 

Posterior  Bow 

Unknown    serum,    1 

drop    (active) 
Complement,  2  units 
1  c.c.  1%  corpuscle 
suspension 

Positive     serum,     1 

drop 
Complement,  2  units 
1  c.c.  1%  corpuscle 

suspension 

Normal      serum,      1 

drop 
Complement,  2  units 
1  c.c.  1%  corpuscle 

suspension 

37°    C.    for 
om  tempera 
sufficient  ai 
ibation. 

Anterior  Bow 

Anterior  Bow 

Anterior  Bow 

at 

it  ro 
bath 

inei 

Unknown    serum,    1 
drop    (active) 

Complement,  2  units 

1  c.c.  1%  corpuscle 
suspension 

Antigen,  1  unit 

Positive     serum,     1 

drop 
Complement,  2  units 
1  c.c.  1%  corpuscle 

suspension 
Antigen.  1  unit 

Normal      serum,      1 

drop 
Complement,  2  units 
1  c.c.  1%  corpuscle 

suspension 
Antigen,  1  unit 

Incubation 
longer,  then  t 
hour  in  watei' 
hour  for  first 

Hecht-Weinberg's  Modification." — In  this  modification 
the  natural  amboceptor  present  in  human  blood  serum  to 
sheep's  corpuscles  is  used  for  the  amboceptor,  and  for 
complement,  that  present  in  the  patient's  blood  serum  is 
utilized.  In  other  words,  the  serum  to  be  tested  furnishes 
both  amboceptor  and  complement.  Therefore,  the  blood 
serum  must  not  be  inactivated  before  use  and  should  be 
used  as  soon  as  possible  after  the  specimen  is  taken.  The 
modification  is  claimed  to  give  more  delicate  results  than 
the  usual  Wassermann  test  but  the  writer  does  not  believe 
that  it  should  be  used  generally,  owing  to  the  errors  that 
may  occur  through  false  positive  reactions  occurring  with 
the  usual  antigens  employed  and  because  the  amount  of 
natural  amboceptor  to  sheep  erythrocytes  is  such  a  vari- 


108  THE   WASSERMANN   TEST 

able  quantity  in  hiunan  blood,  being  excessive  in  some  in- 
stances and  practically  absent  in  others.  The  acetone  in- 
soluble antigen  of  Nognchi  should  be  used  with  this  modi- 
fication. 

Gradwohl's  Modification  of  the  Hecht-Weinberg  Modifi- 
cation.— GrradwohP*  has  greatly  improved  the  technic  of 
the  Hecht-Weinberg  modification  of  the  Wassermann  test 
by  ascertaining  the  hemolytic  strength  of  each  serum  to 
be  tested  to  sheep  corpuscles ;  i.  e.,  the  hemolytic  index  of 
each  serum  to  sheep  corpuscles  is  worked  out  before  the 
serum  is  finally  tested.  GradwohP^  thus  describes  his 
technic : 

''Place  in  a  rack  fourteen  small  test  tubes.  The  first 
ten  of  these  tubes  are  used  to  determine  the  hemolytic  in- 
dex of  the  suspected  blood.  By  this  I  mean  the  exact 
amount  of  hemolytic  amboceptor  present  in  the  given 
blood  serum.  The  last  four  tubes  are  used  in  the  actual 
test.  Add  0.1  c.c.  of  fresh,  unheated,  patient's  blood  serum 
to  each  of  the  first  ten  tubes.  Then  add  decreasing 
amounts  of  normal  salt  solution  to  these  tubes,  beginning 
with  1  c.c,  then  0.9,  0.8,  0.7,  0.6,  0.5,  0.4,  0.3,  0.2  and  0.1  c.c. 
to  the  succeeding  nine  tubes.  Next  add  increasing  amounts 
of  fresh  5  per  cent  suspension  of  sheep's  corpuscles,  start- 
ing mth  0.1  c.c.  and  ending  with  1  c.c.  Place  the  rack  in 
the  water-bath  for  one-half  hour.  The  tube  which  last 
shows  complete  hemolysis  constitutes  the  'hemolytic  in- 
dex;' if  it  is  tube  4,  the  index  is  4,  because  this  tube  had 
received  0.4  c.c.  of  sheep's  corpuscles  and  therefore  we 
have  obtained  an  idea  as  to  how  much  sheep's  blood  is  to 
be  added  to  the  last  four  tubes.  The  first  three  of  these 
tubes  (11,  12,  and  13)  constitute  the  tubes  for  the  actual 
test,  Avhile  the  last  tube  in  the  rack  (14)  serves  as  the  se- 
rum control  tube.  Tubes  11,  12,  and  13  receive,  therefore, 
the  patient's  serum,  the  proper  amount  of  sheep's  corpus- 
cles, dependent  on  hemolytic  index,  rising  strengths  of  an- 
tigen, but  no  complement  and  no  amboceptor.  Tube  14  re- 
ceives only  sheep  corpuscles,  but  no  antigen." 


MODIFICATIONS   OE   THE   WASSEKMAFF   TEST  109 

"In  my  technic  I  use  0.1  c.c.  of  a  diluted  antigen,  deter- 
mined by  titration,  in  tube  11,  0.15  c.c.  antigen  in  tube  12, 
and  0.2  c.c.  in  tube  13.  In  order  to  equalize  the  volume  of 
fluid  in  these  tubes,  I  add  0.2  c.c.  normal  saline  to  tube  11, 
0.15  c.c.  to  tube  12,  0.1  c.c.  to  tube  13,  and  0.3  c.c.  to  tube  14. 
The  tubes  are  then  agitated  and  placed  in  the  water-bath 
for  half  an  hour.  These  last  four  tubes  are  filled  at  the 
time  I  make  the  additions  to  the  first  ten  and  are  left  with 
them  in  the  water-bath  for  one-half  hour  for  fixation  of 
complement,  the  rack  is  then  taken  out  and  the  hemolytic 
index  computed.  If  the  index  is  low,  say  from  1 :  4,  I  add 
0.1  c.c.  of  sheep's  blood  to  the  last  four  tubes.  If  the  in- 
dex is  between  5  and  7,  I  use  0.15  c.c.  sheep's  blood  to  the 
last  four  tubes ;  if  between  8  and  10,  I  add  0.2  c.c. ;  if  the 
index  is  over  10,  I  rack  up  ten  more  tubes  and  repeat  the 
titration  of  the  natural  complement  and  amboceptor,  then 
I  estimate  that ;  if  between  11  and  15,  I  use  0.25  c.c. ;  if 
between  15  and  18,  I  use  0.3  c.c;  and  if  between  18  and 
20,  I  use  0.35  c.c.  If  the  patient's  serum  has  an  index  be- 
low 2,  I  regard  the  reaction  of  doubtful  value.  If  it  is 
above  2,  I  regard  it  as  absolute.  The  reaction  is  read  off 
exactly  as  in  the  "VYassermann,  that  is,  inhibition  or  non- 
inhibition  of  hemolysis.  If  the  amount  of  complement  or 
amboceptor  is  very  low,  we  can  add  the  proper  amount  of 
guinea  pig's  serum  or  rabbit's  immune  serum,  ascertained 
by  preliminary  titration. ' ' 

Gradwohl  claims  that  this  modification,  in  his  hands,  is 
superior  to  the  Wassermann  reaction,  giving  15  to  20  per 
cent  more  positive  reactions  than  the  latter,  but  the  writer 
believes  that  it  is  so  cumbersome  that  it  could  not  be  used 
in  laboratories  where  scores  and,  sometimes,  hundreds  of 
tests  are  being  made  each  day,  as  in  army  practice,  and 
that  the  fact  that  false  positives  have  been  repeatedly  re- 
ported by  different  observers  who  have  used  the  test  mil- 
itates greatly  against  its  value.    It  is  an  excellent  control 


110  THE   WASSEEMANN   TEST 

of  the  Wassermann  test  but  should  not  replace  it  in  gen- 
eral laboratory  practice. 

Kolmer's  Modifications. — Four  different  methods  for 
performing  the  Wassermann  reaction  are  recommended  by 
Kolmer.^''  The  first  method  is  exactly  similar  in  technic  to 
the  original  Wassermann,  except  that  an  alcoholic  extract 
of  syphilitic  liver  is  used  instead  of  an  aqueous  extract. 
In  the  second  method  the  technic  is  the  same  except  that 
three  different  antigens  are  used  with  each  blood  serum 
tested  and  the  amounts  of  each  reagent  are  reduced  one- 
half.  Kolmer  strongly  recommends  this  method  as  being 
"simple,  accurate,  and  reliable."  The  three  antigens  used 
are :  1.  A  cholesterinized  alcoholic  extract  of  human  heart ; 
2,  alcoholic  extract  of  syphilitic  liver;  3,  acetone  insoluble 
lipoids.  It  will  thus  be  noted  that  four  test  tubes  are  re- 
quired for  each  serum  tested,  one  as  control,  and  three 
for  the  antigen  tubes.  As  regards  the  results,  Kolmer 
says : 

"With  strongly  positive  serums  there  is  complete  inhi- 
bition of  hemolysis  with  all  three  antigens.  *  *  *  In  from 
15  to  20  per  cent  of  cases  the  cholesterinized  extract  shows 
a  50  per  cent  or  more  inhibition  of  hemolysis,  Avhereas  with 
the  other  two  antigens  the  reactions  are  negative.  In  our 
experience  the  majority  of  such  serums  were  taken  from 
patients  giving  a  frank  history  of  syphilis  of  many  years' 
standing,  and  from  known  cases  undergoing  treatment,  fur- 
ther therapy  being  indicated  until  the  reaction  finally  be- 
comes negative  when  cholesterinized  extracts  are  used.  In 
a  small  proportion  of  cases  a  feebly  positive  reaction  of 
25  per  cent  or  even  less  of  inhibition  of  hemolysis  may  be 
found  with  the  cholesterinized  extract  alone.  Many  of  these 
reactions  occur  with  serums  of  treated  cases  of  syphilis ;  on 
the  other  hand  a  similar  reaction  may  occur  with  about  5 
per  cent  of  normal  serums,  so  that  if  the  history  and  clin- 
ical conditions  are  clearly  negative,  a  slight  degree  of  in- 
hibition of  hemolj^sis  (5  to  10  per  cent)  with  the  choles- 


MODIFICATIONS   OF   THE   WASSERMANN   TEST  111 

terinized  extract  and  marked  hemolysis  with  the  other  two 
antigens  may  be  interpreted  as  a  negative  reaction. ' ' 

The  writer  has  quoted  Kohner  regarding  his  results  with 
cholesterinized  extracts  for  antigens  because  his  experi- 
ence has  been  exactly  similar  and  for  this  reason  the  writer 
firmly  believes  in  the  value  of  such  extracts  in  the  diagno- 
sis of  syphilis  with  the  Wassermann  test  and  in  the  control 
of  the  treatment  of  the  disease.  Properly  titrated  and  con- 
trolled, and  with  experience  gained  by  using  them,  so  that 
a  proper  interpretation  may  be  placed  upon  the  results  ob- 
tained, cholesterinized  antigenic  extracts  are  perfectly  safe 
and  furnish  a  much  greater  amount  of  information  regard- 
ing the  existence  of  a  syphilitic  infection  than  do  other 
antigenic  extracts. 

Kolmer's  third  method  is  devised  in  order  to  measure 
the  amount  of  syphilitic  antibody  in  the  patient's  blood  se- 
rum. He  employs  only  one  antigen,  either  an  alcoholic 
extract  of  syphilitic  liver  or  the  acetone  insoluble  lipoid 
antigen.  The  technic  is  about  the  same  as  in  the  original 
Wassermann  but  eight  tubes  are  used  for  each  patient's 
serum,  increasing  amounts  of  the  serum,  diluted  1 :10  being 
placed  in  them,  together  with  the  unit  of  antigen  deter- 
mined by  titration,  only  one  antigen  being  emploj^ed.  The 
fourth  method  is  somewhat  similar,  but  in  this  the  amount 
of  syphilitic  antibody  in  the  patient's  blood  serum  is  meas- 
ured by  the  number  of  hemolytic  doses  of  complement  fixed 
by  a  constant  amount  of  antigen. 

As  routine  methods,  where  many  tests  are  being  per- 
formed, the  third  and  fourth  methods  of  Kolmer  are  not 
practical,  as  they  consume  too  much  time  and  material. 
The  second  method  is  an  excellent  one,  but  it  appears  to 
the  writer  that  practically  the  same  results  are  obtained  by 
using  two  antigens,  instead  of  three,  as  recommended  in 
his  modification.  The  writer  also  believes,  as  already 
stated,  that  a  human  hemolytic  system  is  preferable  to  the 
sheep  system,  which  is  used  in  all  of  Kolmer's  methods, 
owing  to  the  possibility  of  the  natural  antisheep  ambocep- 


112  THE   WASSERMANN   TEST 

tor  present  in  nearly  all  human  blood  serum  interfering 
with  the  delicacy  of  the  test. 

Despite  all  the  claims  that  have  been  made  that  the  pres- 
ence of  antisheep  amboceptor  in  human  serum  is  a  negligi- 
ble factor  in  the  Wassermann  test,  the  fact  remains  that 
in  some  blood  sera  there  is  so  much  of  this  amboceptor 
present  that  no  dependence  can  be  placed  upon  the  results 
of  a  Wassermann  test  with  them  unless  it  is  first  removed, 
and  it  is  certainly  logical  to  believe  that,  in  some  cases, 
this  amboceptor  when  combined  with  the  amboceptor 
added,  would  render  a  reaction  negative  that,  with  the  hu- 
man hemolytic  system,  would  be  positive.  This  factor 
would  frequently  be  of  very  great  importance  in  any 
method  of  performing  the  test  in  which  titrations  of  the 
amount  of  syphilitic  antibody  in  the  patient's  serum  were 
used. 

The  modifications  of  Tschernogubow"  which  utilize  the 
natural  complement  and  amboceptor  against  guinea  pig 
corpuscles  present  in  human  serum;  of  Detre  and  Bre- 
zovsky,^^  in  which  an  antihorse  hemolytic  system  is  em- 
ployed ^dth  rabbit  complement ;  of  Margarette  Stern^^  who 
used  fresh,  active  blood  serum  and  the  complement  present 
in  the  blood  serum  tested;  of  Bauer ,^°  who  makes  use  of 
the  antisheep  amboceptor  present  in  human  blood  serum; 
of  Browning  and  McKenzie,®^  who  use  an  antiox  hemolytic 
system  and  a  quantitative  technic ;  and  of  Thompson,^^  who 
uses  the  complement  present  in  the  fresh  blood  serum  of 
the  patient,  an  antihuman  hemolytic  S3^stem,  and  the 
acetone  insoluble  lipoid  antigen  of  Noguchi  have  all  been 
recommended,  from  time  to  time,  but  none  of  them  appear 
to  be  as  reliable  as  the  original  Wassermann  technic,  the 
modifications  recommended  by  Noguchi  and  Kolmer,  or 
the  writer's  modification. 

The  Effect  of  Low  Temperatures  upon  the  Results  of 
the  Wassermann  Test. — Quite  recently  several  observers 
have  called  attention  to  the  effect  of  incubating  the  com- 


MODIFICATIONS   OF   THE   WASSEEMANN   TEST  113 

plement,  antigen,  and  patient's  blood  serum  at  ice  box  tem- 
perature, claiming  that  more  delicate  results  are  obtained 
than  when  this  incubation  is  made  in  the  water-bath  at 
37°  C.  Zinsser"^  states  that  in  his  laboratory  two  series  of 
tests  were  run,  one  A\dth  the  first  incubation  at  37.5°  C.  for 
30  minutes,  the  other  with  the  first  incubation  in  the  ice 
box  at  8  to  10°  C,  for  three  hours,  the  same  blood  sera 
being  used  in  each  series.  The  results  showed  15  more 
positive  reactions  in  the  series  incubated  at  ice  box  tem- 
perature, than  in  that  incubated  in  the  water-bath,  and  of 
these  cases  7  were  unquestionably  syphilitic,  two  were 
treated  syphilitics,  and  four  were  probabl}^  syphilitic. 

In  a  recent  valuable  contribution.  Smith  and  MacNeaP* 
compared  the  results  obtained  with  different  antigens  and 
different  temperatures  of  incubation  in  the  Wassermann 
test.  They  experimented  with  three  antigens  as  follows: 
1.  A  cholesterinized  alcoholic  extract  of  beef  heart.  2.  A 
plain  alcoholic  extract  of  beef  heart.  3.  An  acetone  insolu- 
ble extract  of  beef  heart.  Two  series  of  tests  were  made, 
with  the  same  blood  sera,  one  in  which  the  first  incubation, 
with  each  antigen,  was  at  37°  C.  and  one  in  which  the  first 
incubation  was  for  4  hours  in  the  ice  box  at  8°  C,  with  each 
antigen.  Over  500  different  sera  were  tested  and  their  con- 
clusions are  important.  They  found  that  the  cholesterin- 
ized antigen,  with  incubation  at  8°  C.  for  4  hours  proved  a 
more  sensitive  test  for  syphilis  than  any  of  the  other 
methods  tried  but  that  it  was  apt  to  give  nonspecific  com- 
plement fixation,  and,  therefore,  if  used  alone  it  could  not 
be  depended  upon  in  diagnosis.  The  simple  extract  antigen 
(alcoholic  extract  of  beef  heart),  with  the  first  incubation 
for  4  hours  at  8°  C.  in  the  ice  box,  gave  more  sensitive  re- 
sults than  the  cholesterinized  extract  at  37°  C.  and  in  these 
experiments  did  not  give  any  false  positive  results.  The 
acetone  insoluble  extract  was  found  less  sensitive  either 
at  37°  C.  or  8°  C.  than  the  cholesterinized  extract  and  also 
less  sensitive  than  the  simple  alcoholic  extract  at  8°  C. 


114  THE   WASSERMAITE"   TEST 

It  was  more  sensitive  than  the  simple  extract  at  37°  C, 
and  gave  no  false  positive  results  in  this  series  of  tests. 
From  their  results  it  is  evident  that  the  cholesterinized  ex- 
tract at  8°  C.  gave  the  highest  percentage  of  positive  re- 
sults, but  sometimes  gave  a  false  positive  reaction,  while 
the  simple  alcoholic  extract  gave  the  best  practical  results, 
when  the  first  incubation  was  made  at  8°  C,  as  it  gave 
better  results  than  the  cholesterinized  extract  at  37°  C, 
and  did  not  give  any  false  reactions. 

The  subject  of  the  ice  box  temperature  for  the  first  in- 
cubation in  the  Wassermann  test  deserves  further  study, 
and,  if  these  observations  are  confirmed,  this  temperature 
should  be  adopted  in  practice.  If  it  is  found  reliable  it 
will  probably  be  best  to  use  simple  alcoholic  extracts  for 
antigen  instead  of  cholesterinized  extracts,  but  more  work 
should  be  done  upon  the  subject  before  the  method  is  gen- 
erally adopted. 

The  Titration  of  the  Strength  of  the  Wassermann  Reac- 
tion.— In  using  the  Wassermann  test  as  a  control  of  the 
efficiency  of  treatment,  it  is  often  advisable  to  test  the 
strength  of  the  reaction,  from  time  to  time,  especially  if 
sera  show  a  double-plus  (four-plus  reaction)  repeatedly, 
although  intensive  treatment  is  being  administered.  In 
such  cases,  the  physician  can  not  be  sure  that  the  treat- 
ment is  having  any  effect  unless  the  blood  serum  is  titrated 
as  to  its  fixing  or  absorbing  power  of  complement  in  the 
presence  of  suitable  antigen.  This  is  done  by  diluting  the 
blood  serum  in  the  following  manner,  as  recommended  by 
Vedder  f^  Six  tubes  are  used  in  addition  to  the  usual  num- 
ber used  in  the  Wassermann  test  as  recommended  by  the 
writer.  In  the  first  tube  is  placed  1.9  c.c.  of  normal  salt 
solution,  and  in  each  of  the  other  tubes  1  c.c.  of  normal 
salt  solution.  In  the  first  tube  is  placed  0.1  c.c.  of  the 
serum  to  be  titrated  and  the  tube  is  thoroughly  shaken. 
One  cubic  centimeter  is  taken  from  this  tube  and  placed 
in  the  second  tube,  which  is  shaken,  and  the  same  amount 
is  carried  to  the  third  tube,  and  the  operation  repeated 


MODIFICATIONS   OF   THE   WASSEEMANF   TEST 


115 


until  the  sixth  tube  is  reached,  from  which,  after  it  is 
shaken,  1  c.c.  is  discarded.  After  the  dilutions  are  thus 
made  each  tube  will  contain  1  c.c.  of  fluid  and  the  dilutions 
of  the  blood  serum  will  be  as  follows:  Tube  1,  0.05  c.c; 
Tube  2,  0.025  c.c;  Tube  3,  0.012  c.c;  Tube  4,  0.006  cc; 
Tube  5,  0.003  c.c. ;  and  Tube  6,  0.0015  c.c 

The  test  is  then  made  in  the  usual  manner  with  all  of 
the  dilutions  and  the  results  recorded  as  in  Table  XXII. 


Table  XXII 
Results  op  Titration  of  Blood  Serum  of 


DATE  OF  TEST 

DILUTIONS 

0.1  c.c. 

0.05 

0.025 

0.012 

0.006 

0.003 

0.0015 

Previous  Eesult. 

+  + 

with  0.05  c.c. 

+  + 

+ 

+  - 

- 

- 

- 

- 

This  method  of  titrating  the  blood  serum  of  patients 
under  treatment  is  recommended  in  all  cases  in  which  per- 
sistent double-plus  (four-plus)  reactions  are  reported,  as 
it  is  the  only  way  in  which  the  physician  can  be  snre  that 
the  particular  method  of  treatment  being  pursued  is  prov- 
ing efficient.  In  many  instances  the  titration  will  demon- 
strate that  the  smaller  amounts  of  serum  become  progres- 
sively negative,  thus  proving  the  efficiency  of  the  treatment, 
although  the  usual  amount  of  serum  tested,  i.  e.,  0.1  cc, 
may  give  a  positive  reaction. 


CHAPTER  VI 

COMPLEMENT  FIXATION  IN  SYPHILIS  WITH 

ANTIGENS  PREPARED  FROM  PURE 

CULTURES  OF  TREPONEMA 

PALLIDUM 

The  success  attending  Noguchi's  efforts  to  obtain  Tre- 
ponema pallidum  in  pure  culture  lead  to  the  hope  that  an- 
tigens prepared  from  such  cultures  would  be  effective  in 
the  Wassermann  test  and  that  in  this  manner  a  truly  spe- 
cific antigen  would  be  obtained  and  the  reaction  become 
a  true  antigen-antibody  reaction.  It  is  obvious  that  if 
such  a  specific  antigen  could  be  obtained  the  value  of  the 
Wassermann  test,  great  as  it  is  at  present,  would  become 
still  greater,  as  the  positive  reactions  frequently  observed 
in  yaws,  tubercular  leprosy,  and  Avith  less  frequency,  in 
malaria  and  trypanosomiasis,  would  be  eliminated.  The 
subject  has  already  been  briefly  discussed  but  it  is  thought 
of  enough  importance  to  consider  somewhat  more  exten- 
sively in  this  chapter. 

Schereschewsky,  in  1909,®^  was  the  first  to  attempt  to 
secure  an  antigen  from  cultures  of  Treponema  pallidum. 
Unfortunately,  his  culture  was  an  impure  one  and  for  this 
reason  his  results  were  not  conclusive.  He  was  able  to 
obtain  complement  fixation  with  syphilitic  serum,  with  his 
antigen,  but  also  obtained  positive  results  with  some  of  the 
control  antigens  with  which  he  worked.  It  is  now  gen- 
erally admitted  that  he  was  not  working  with  cultures  of 
Treponema  pallidum  at  all  but  with  Treponema  microden- 
tium,  and,  as  will  be  sho^^m  later,  his  positive  results  were 
in  the  nature  of  a  group  reaction,  as  the  writer  and  Nichols 
have  shown  that  this  organism  "will  react  positively  with 
syphilitic   serum,   if  used   as   an   antigen.     In   addition, 

116 


COMPLEMENT   EIXATIOjST   IIST    SYPHILIS  117 

Schereschewsky's  cultures  were  contaminated  with  bac- 
teria and  it  is  possible  that  some  of  his  positive  results 
were  due  to  this  fact,  for,  as  already  noted,  certain  bacteria, 
when  growing  in  normal  blood  serum,  produce  substances 
which  give  a  positive  reaction  with  the  Wassermann  test, 
and  it  is  not  improbable  that  bacteria  growing  in  the  horse 
serum  medium,  which  he  used  for  his  cultures,  may  have 
produced  lipoidal  substances  which  were  capable  of  giving 
positive  results.  However,  his  work  was  very  suggestive 
and,  without  doubt,  stimulated  research  along  these  lines. 

In  1912,  Noguchi,''^  who  had  succeeded  in  isolating  and 
growing  Treponema  pallidum  in  pure  culture,  published 
the  results  of  his  work  upon  complement  fixation  in  syph- 
ilis with  an  antigen  prepared  from  such  cultures.  His  an- 
tigen was  an  aqueous  extract  of  Treponema  pallidum,  and 
he  also  worked  Avith  an  emulsion  of  the  organisms  obtained 
from  the  testicles  of  infected  rabbits.  His  researches  dem- 
onstrated beyond  doubt  that,  in  certain  cases  of  syphilitic 
infection,  it  is  possible  to  obtain  complement  fixation  mth 
antigens  prepared  from  a  pure  culture  of  Treponema  palli- 
dum and  that,  in  such  instances,  the  reaction  is  indeed  a 
true  antigen-antibody  reaction. 

Noguchi  found  that,  in  cases  of  syphilis  that  had  been 
treated  or  in  which  the  disease  was  latent,  he  often  ob- 
tained a  positive  reaction  with  the  culture  antigen  when 
the  ordinary  antigens  used  in  the  Wassermann  test  gave 
a  negative  result,  and  that  the  culture  antigen  very  rarely 
gave  a  positive  result  in  primary  and  secondary  cases  of 
the  disease.  Therefore,  he  concludes  that  the  reaction  with 
the  pallidum  antigen  may  be  regarded  as  an  index  of  the 
resistance  of  the  patient  to  the  infection,  stating  ''We  have 
in  the  Wassermann  reaction  a  fair  measure  of  the  ac- 
tivity of  the  infecting  agent,  and  now  we  will  have  in  the 
pallida  fixation  reaction  a  gauge  for  the  defensive  activity 
of  the  infected  host." 

Noguchi 's  conclusions  regarding  his  work  with  the  pal- 
lidum antigen  are  as  follows: 


118  THE   WASSERMANlsr   TEST 

1.  The  Wassermann  reaction  is  caused  by  the  lipotropic 
substances  in  sera  but  not  by  the  antibodies  which  combine 
specifically  with  the  pallida  antigen. 

2.  The  fixation  produced  by  the  culture  pallida  antigen 
with  certain  syphilitic  sera  is  caused  by  the  specific  anti- 
bodies contained  in  the  latter  and  may  constitute  a  specific 
diagnostic  method  for  syphilis. 

3.  The  fixation  caused  by  testicular  extracts  behaves  like 
the  culture  pallida  antigen  in  the  majority  of  cases,  but 
when  the  sera  (syphilitic  or  leprous)  contains  abundant 
lipotropic  substances,  they  may  give  a  Wassermann  re- 
action as  well,  which  is  not  the  case  with  the  culture  pallida 
antigen. 

4.  In  the  serum  of  rabbits  with  active  syphilitic  orchitis 
there  is  no  indication  of  the  presence  of  a  sufficient  amount 
of  antibodies  for  the  pallida  antigen,  although  it  gives  a 
strong  Wassermann  reaction. 

In  1912,  the  writer  and  Major  Nichols  published  the 
results  of  some  work  upon  specific  antigens  in  complement 
fixation  with  syphilitic  serum,®®  which  differed  somewhat 
from  those  of  Noguchi,  chiefly  because  of  the  nature  of 
the  antigen  employed.  In  his  work  Noguchi  used  aqueous 
extracts  of  Treponema  pallidum,  Avhile  in  our  work  alco- 
holic extracts  were  employed,  Noguchi  stated  that  it  was 
impossible  to  extract  the  antigenic  properties  of  the  or- 
ganism by  alcohol  and  that  only  aqueous  extracts  contained 
them,  but  our  results  showed  that  alcoholic  extracts  were 
efficient,  although  they  were  not  as  efficient  as  aqueous  ex- 
tracts, and  our  work  has  since  been  confirmed  by  other  au- 
thorities. It  is  true  that  alcoholic  extracts  owe  a  part  of 
their  activity  to  other  substances  than  the  specific  com- 
plement-fixing substance  of  Treponema  pallidum  which  is 
best  extracted  in  water,  but  it  is  also  true  that  the  alco- 
holic extracts  do  contain  this  specific  substance,  as  the  re- 
sults obtained  can  be  explained  in  no  other  way,  and  dif- 
fer so  greatly  from  those  obtained  with  ordinary  alcoholic 
antigens  used  in  the  Wassermann  test. 


COMPLEMEIsrT   FIXATION   IN   SYPHILIS  119 

In  our  experiments  we  tested  51  syphilitic  sera  and  54 
nonsyphilitic  sera.  Of  the  latter,  38  were  from  patients 
suffering  from  diseases  other  than  syphilis  and  16  were 
from  normal  individuals.  Eleven  rabbit  sera  were  also 
tested.  The  sera  were  all  inactivated  by  heating  them  at 
55-56°  C.  for  half  an  hour  before  testing. 

The  stock  antigen  used  was  an  alcoholic  extract  of  foetal 
syphilitic  liver  and  the  culture  antigens  M^ere  prepared 
from  cultures  kindly  sent  us  by  Dr.  Noguchi.  Alcoholic 
extracts  were  made  of  the  cultures,  filtered,  the  filtrate 
evaporated  to  one-third  its  original  volume  and  titrated  in 
a  dilution  of  1-10  with  normal  salt  solution.  Antigens 
were  thus  prepared  from  cultures  of  Treponema  pallidum, 
Treponema  pertenue,  and  Treponema  microdentium.  The 
test  was  performed  in  the  same  manner  as  that  recoiri- 
mended  in  this  work  by  the  writer  for  the  Wassermann 
test. 

As  a  control  of  the  culture  antigens  used  an  alcoholic  ex- 
tract of  the  medium  employed  in  growing  the  treponemas 
was  prepared  in  the  same  manner  as  the  culture  antigens. 

Results  with  Normal  Sera. — The  blood  serum  of  16  nor- 
mal individuals  was  tested  with  the  stock  antigen  and  with 
pallidum,  pertenue,  and  microdentium  antigen,  and  all  gave 
a  negative  result.  Not  a  trace  of  a  reaction  occurred  in 
any  of  these  tests  so  that  it  is  evident  that  none  of  the  an- 
tigens emploj^ed  reacted  with  normal  blood  serum. 

Results  with  Serum  from  Other  Diseases  than  Syph- 
ilis.— The  blood  serum  of  38  patients  suffering  from  dis- 
eases other  than  syphilis  was  tested  with  the  same  an- 
tigens and  was  negative  in  every  instance,  except  that  a 
plus  reaction  was  obtained  with  the  microdentium  antigen 
in  a  case  diagnosed  as  arthritis.  Syphilis  could  not  be 
eliminated  in  this  case  but  the  stock  syphilitic  antigen  gave 
a  negative  result  with  the  serum. 

Results  with  Serum  from  Syphilitic  Patients. — The  blood 
sera  of  51  patients  suffering  from  syphilis  were  tested  and 
Table  XXIII  shows  the  results  obtained.     In  recording 


120 


THE   WASSERMANN   TEST 


these  tests  the  sign,  ++,  means  absolute  inhibition  of  hem- 
olysis; the  sign,  +,  at  least  50  per  cent  of  inhibition;  and 
the  sign,  +-,  weaker  degrees  of  inhibition.  The  sign,  -, 
indicates  total  lack  of  inhibition,  while  the  sign  0  indicates 
that  no  test  was  made. 

Table  XXIII 

Complement  Fixation  in  Syphilis  with  Stock  Antigen  and  Specific 

Antigens  in  Forty  Infections* 


T.  PAL- 

T. PER- 

T.  MICRO- 

CONTROL 

SERUM 

stock 

LIDUM 

TENUE 

DENTIUM 

ANTIGEN 

REMARKS 

NO. 

ANTIGEN 

ANTIGEN 

ANTIGEN 

ANTIGEN 

1 

+  + 

+  + 

0 

0 

_ 

Secondary,  early 

2 

+  + 

+ 

+  - 

0 

_ 

Primary 

3 

+  + 

+  - 

+  - 

0 

_ 

Secondary 

4 

+  + 

_ 

0 

0 

0 

Latent,  18  years 

5 

+  + 

+ 

+ 

+ 

_ 

Latent,  4  years 

6 

+  + 

_ 

+  - 

_ 

_ 

Latent,  3  years 

7 

+  + 

_ 

_ 

0 

_ 

Tertiary,  10  years 

8 

+  + 

_ 

0 

0 

_ 

Secondary,  9  months 

9 

+ 

+ 

0 

0 

0 

Secondary,  early 

10 

+ 

0 

0 

0 

_ 

Primary 

11 

+ 

+  + 

0 

0 

_ 

Primary,  2  weeks 

12 

+    ■ 

+ 

0 

0 

0 

Latent,  2  years 

13 

+ 

_ 

0 

+  - 

0 

Primary 

14 

+ 

_ 

0 

+ 

- 

Primary 

15 

+ 

+ 

0 

+ 

- 

Secondary,  early 

16 

+ 

+ 

- 

- 

- 

Secondary,  1  year 

17 

+ 

_ 

0 

+ 

- 

Tertiary 

18 

+ 

+ 

+  - 

+ 

- 

Secondary 

19 

+ 

+  + 

0 

+ 

- 

Tertiary 

20 

+ 

+  - 

+  - 

+  - 

- 

Latent,  duration  un- 
known 

21 

+  - 

+  - 

0 

0 

_ 

Latent,  3  years 

22 

+  - 

+ 

0 

0 

- 

Primary,  3  weeks 

23 

+  - 

+  - 

0 

0 

0 

Secondary,  early 

24 

+  - 

- 

- 

- 

_ 

Primary,  2  weeks 

25 

+  - 

- 

- 

0 

- 

Epilepsy 

26 

+  - 

+  - 

0 

+- 

- 

Arthritis,  syphilitic 

27 

+  - 

+  - 

0 

+  - 

0 

Latent,  2  years 

28 

+  - 

+  - 

0 

+  - 

_ 

Latent,  2  years 

29 

+  - 

+ 

0 

+ 

_ 

Latent,  1  year 

30 

+  - 

+  - 

- 

+  - 

- 

Secondary,  1  year 

31 

+  - 

+  + 

+  + 

+ 

- 

Latent,  duration  un- 
known 

32 

+  - 

+  - 

0 

+ 

_ 

Latent,  3  years 

33 

+  - 

+  - 

+  - 

- 

- 

Secondary,  3  months 

34 

+  - 

+ 

+  - 

+  - 

_ 

Latent,  4  years 

35 

+  - 

+  - 

_ 

+  - 

_ 

Latent,  6  years 

36 

+  - 

+  - 

+  - 

- 

- 

Secondary,  early 

37 

_ 

+  - 

_ 

+  - 

_ 

Latent,  2  years 

38 

- 

+ 

+ 

+  - 

- 

Latent,  1%  years 

39 

_ 

+  - 

_ 

_ 

_ 

Latent,  6  years 

40 

- 

+  - 

- 

0 

0 

Primary,  2  weeks 

*Each  serum  tested  was  properly  controlled  as  is  usual  in  the  Wassermann  test. 


COMPLEMENT   FIXATION   IF   SYPHILIS  121 

It  will  be  noted  that  no  case  reacted  with  the  control 
culture  medium  antigen  and  that  of  the  36  cases  giving 
any  reaction  at  all  with  the  stock  extract  antigen,  no  less 
than  twenty-seven,  or  75  per  cent  reacted  also  with  the 
pallidum  antigen,  but  the  reactions  were,  as  a  rule,  much 
weaker  with  the  pallidum  antigen  than  with  the  stock  an- 
tigen. However,  there  were  instances  in  which  the  pal- 
lidum antigen  gave  a  stronger  reaction  than  the  stock  an- 
tigen, as  illustrated  in  serums  11  and  19,  while  in  serum 
38  the  stock  antigen  gave  a  negative  result  and  the  pal- 
lidum antigen  a  plus  reaction. 

As  regards  the  results  given  with  the  antigen  prepared 
from  th^  pure  cultures  of  Treponema  pallidum,  17  of  the 
sera  gave  the  same  result  as  with  the  stock  antigen,  four 
gave  weaker  reactions,  six  gave  stronger  reactions,  and 
four  gave  positive  results  in  sera  in  which  the  stock  an- 
tigen gave  a  negative  result.  However,  the  pallidum  an- 
tigen gave  seven  negative  reactions  in  twenty  sera  that 
gave  absolute  inhibition  of  hemolysis  with  the  stock  an- 
tigen, (not  shown  in  table)  thus  proving  that  the  pallidum 
antigen  is  untrustworthy  in  the  diagnosis  of  syphilis. 

Of  the  various  sj^philitic  sera  tested,  fifteen  were  tested 
with  all  three  antigens,  and  the  results  are  valuable  be- 
cause they  indicate  the  group  nature  of  the  complement 
fixation  reaction  obtained  with  these  alcoholic  extracts  of 
Treponema  pallidum,  Treponema  pertenue,  and  Treponema, 
microdentium.    Table  XXIV  gives  the  results  obtained : 

In  four  of  the  cases,  the  results  were  the  same  with  all 
of  the  culture  antigens  but  in  the  remaining  cases  they 
varied,  the  pallidum  antigen  giving  results  more  like  those 
of  the  stock  antigen,  while  the  pertenue  and  microdentium 
antigens  gave  quite  similar  results,  although  those  of  the 
microdentium  approached  nearer  to  those  of  the  pallidum 
than  did  the  pertenue. 

Unlike  Noguchi,  we  obtained  positive  results  not  only  in 
latent  syphilitic  infections  but  also  in  primary  and  second- 


122 


THE   WASSERMANF   TEST 


Table  XXIV 
Complement  Fixation  in  Syphilitic  Serum  with  Cultuke  Antigens 


SERUM 
NO. 


1 

2 

3 

4 

5 

6 

7 

8 

9 

10 

11 

12 

13 

14 

15 


STOCK 

antigen 


+  + 
+  + 

+ 
+  - 

+ 

+ 
+  - 
+  - 
+  - 
+  - 
+  - 


T.  PAL- 
LIDUM 
ANTIGEN 


+ 
+  - 

+ 

+ 
+  - 
+  4- 
+  - 

+ 

+  - 
+  - 

+ 
+  - 
+  - 


T.  PER- 

TENUE 
ANTIGEN 


+ 


+  - 


+  - 

+  + 
+  - 
+  - 


T.  MICRO- 
DENTIUM 
ANTIGEN 


+ 
+  - 


+ 

+  - 

+ 

+  - 
+  - 
+  - 
+  - 


0 


CONTROL 
ANTIGEN 


REMARKS 


Latent,  4  years 
Latent,  3  years 
Latent,  duration? 
Primary,  2  weeks 
Secondary,  1  year 
Secondary,  duration? 
Latent,  duration? 
Secondary,  3  months 
Latent,  4  years 
Latent,  6  years 
Latent,  2  years 
Latent,  1%  years 
Latent,  6  years 
Primary,  2  weeks 
Secondary,  1  year 


ary  infections  with  the  culture  antigens.  It  should  be  re- 
membered, in  this  connection,  that  Noguchi  worked  mth 
an  aqueous  extract  of  Treponema  'pallidum  while  we  used 
alcoholic  extracts,  and  thus  the  variation  may  be  explained, 
although  the  writer  does  not  agree  with  Noguchi  that  it  is 
impossible  to  extract  from  this  organism  specific  comple- 
ment-fixing substances  with  alcohol.  Our  results  with  these 
alcoholic  extracts  of  treponemas  are  too  much  at  variance 
with  those  obtained  mth  the  ordinary  alcoholic  extract  of 
foetal  syphilitic  liver  to  make  it  at  all  probable  that  the 
entire  complement-fixing  or  absorbing  power  of  these  cul- 
ture antigens  was  entirely  nonspecific  in  character,  and 
our  experiments  with  rabbit  blood  serum,  which  gave 
identical  results  with  those  of  Noguchi,  are  evidence  of  this 
fact. 

Results  With  Rabbit  Blood  Serum. — ^We  tested  the 
blood  serum  of  two  rabbits  presenting  acute  symptoms  of 
syphilitic  infection;  of  three  that  had  recovered  from  a 
syphilitic  orchitis;  of  two  that  had  recovered  from  yaws; 
and  of  four  normal  rabbits,  and  the  results  are  given  in 
Table  XXV. 

The  important  fact  shown  by  these  experiments  is  that 


COMPLEMENT   EIXATION   IN    SYPHILIS 


123 


Table  XXV 
Complement  Fixation  with  Specific  Treponema  Antigens  in  Rabbit  Sera 


T.  PAL- 

T. PER- 

T.  MICRO- 

RABiBIT 

STOCK 

LIDUM 

TENUE 

DENTIUM 

CONTROL 

REMARKS 

NO. 

ANTIGEN 

ANTIGEN 

ANTIGEN 

ANTIGEN 

ANTIGEN 

1 

+  + 

_ 

_ 

_ 

_ 

Active  lesions 

2 

+  + 

_ 

_ 

_ 

_ 

Active  lesions 

3 

^ 

- 

_ 

_ 

0 

Recovered  from  syphilis 

4 

- 

- 

_ 

- 

0 

Recovered  from  syphilis 

5 

- 

- 

- 

- 

- 

Recovered  from  syphilis 

6 

- 

- 

- 

0 

_ 

Recovered  from  yaws 

7 

- 

- 

- 

- 

_ 

Recovered  from  yaws 

8 

— 

_ 

_ 

0 

_ 

Normal  rabbit 

9 

— 

_ 

_ 

0 

_ 

Normal  rabbit 

10 

— 

_ 

_ 

0 

_ 

Normal  rabbit 

11 

- 

- 

- 

0 

0 

Normal  rabbit 

no  reaction  with  the  culture  antigens  occurred  in  any  of  the 
rahhit  sera,  although  the  two  animals  showing  active  le- 
sions of  syphilis  gave  absolute  inhibition  of  hemolysis 
with  the  stock  antigen.  These  results  are  in  entire  agree- 
ment with  those  of  Noguchi,  who  found  that  his  pallida  an- 
tigen did  not  react  with  rabbit  serum  even  when  the  ani- 
mals presented  active  lesions  of  the  infection.  On  the 
other  hand,  antigens  prepared  by  extracting  with  alcohol 
the  testicles  of  rabbits  presenting  a  syphilitic  orchitis  will 
give  positive  results  with  syphilitic  sera  and  also  with  the 
blood  of  rabbits  injected  with  syphilis,  thus  proving  that 
the  alcoholic  extract  of  cultures  of  Treponema  pallidum 
is  an  entirely  different  antigen  from  that  obtained  from 
the  infected  testicle  of  rabbits  and  that  the  reactions  ob- 
tained with  such  an  extract  are,  at  least,  partly  specific  in 
character.  In  1911,  Nichols  published  some  experiments  of 
ours  upon  complement  fixation  with  alcoholic  antigens 
made  from  the  testicles  of  rabbits  suffering  from  experi- 
mental syphilis  and  yaws,  and  we  found  that  while  such 
antigens  gave  positive  results  Avith  certain  syphilitic  sera, 
they  also  reacted  with  certain  normal  sera,  as  well  as  sera 
from  other  diseases,  and  that  an  antigen  prepared  from 
the  testicles  of  rabbits  inoculated  with  syphilis  give  posi- 
tive results  with  the  sera  of  rabbits  inoculated  with  yaws 


124  THE   WASSERMANlSr   TEST 

and  vice  versa.  In  addition,  we  found  that  alcoholic  an- 
tigens prepared  from  the  testicles  of  normal  rabbits  also 
gave  positive  results,  so  that  we  did  not  consider  that  this 
type  of  antigen  was  at  all  specific  in  character. 

From  our  experiments  with  antigens  prepared  from 
pure  cultures  of  the  treponemas  named  we  reached  the 
following  conclusions: 

1.  Alcoholic  extracts  of  pure  cultures  of  Treponema  pal- 
lidum can  not  be  depended  upon  in  the  diagnosis  of  syph- 
ilitic infection  by  the  complement-fixation  test,  for  many 
cases  of  undoubted  syphilis,  giving  absolute  inhibition  of 
hemolysis  mth  the  stock  antigen,  an  alcoholic  extract  of 
foetal  syphilitic  liver,  gave  a  negative  reaction  "vvith  the 
culture  antigen,  and  the  reactions  obtained  with  the  pal- 
lidum antigen  were  generally  weaker  and  less  conclusive 
than  with  the  stock  antigen. 

2.  The  results  appear  to  prove  that  there  are  specific 
group  reactions,  in  the  way  of  complement  fixation,  or 
absorption,  between  Treponema  pallidum,  Treponema  per- 
tenue  and  Treponema  microdentium,  as  antigens  prepared 
from  pure  cultures  of  these  organisms  may  all  give  comple- 
ment fixation  in  the  presence  of  syphilitic  serum. 

Later  experiments^^  with  antigens  prepared  from  pure 
cultures  of  SpirochcBta  refringens,  Spirillum  cholercB,  and 
Bacillus  typhosus  demonstrated  that,  while  SpirocTiceta  re- 
fringens sometimes  reacted  positively  with  the  sera  tested, 
the  antigens  prepared  from  the  cultures  of  Spirillum  cJiol- 
ercB  and  Bacillus  typhosus  never  give  a  positive  reaction, 
thus  proving  that  the  theory  that  positive  reactions  with 
these  pure  culture  antigens  might  be  due  to  lipoidal  sub- 
stances produced  in  the  media  itself  by  the  growth  of  the 
organisms  is  practically  imtenable,  and  that  there  are 
group  reactions  among  this  class  of  organisms  and  that 
alcoholic  extracts  of  these  organisms  contain  some  specific 
substance  common  to  all,  to  a  greater  or  lesser  extent. 

Since  our  work  a  considerable  number  of  investigators 
have  studied  the  immunological  phenomena  of  cultivated 


COMPLEMENT   FIXATION   IN   SYPHILIS  125 

treponemas  and  spirochetes,  and  onr  results  have  been 
confirmed.  Thus,  Kolmer,  Williams,  and  Laubangh,^" 
working  with  aqueous  and  alcoholic  extracts  of  Treponema 
pallidum  obtained  positive  reactions  in  secondary,  tertiary, 
and  congenital  syphilis.  They  found  that  the  aqueous  ex- 
tracts gave  better  results  than  the  alcoholic  but  that,  with 
both,  the  reactions  were  generally  weaker  than  those  ob- 
tained with  the  ordinary  antigenic  extracts  used  in  the 
Wassermann  test.  They  also  obtained  weak  reactions  with 
control  antigens  made  from  uninoculated  media  and  other 
organisms.  Zinsser,  Hopkins,  and  McBurney"  in  an  ex- 
tended series  of  studies  upon  Treponema  pallidum  and 
syphilis  obtained  specific  agglutination  and  complement 
fixation  with  culture  treponemas  but  conclude  that  their 
work  lends  no  support  to  the  belief  that  either  agglutina- 
tion or  complement  fixation  with  cultures  of  Treponema 
pallidum  will  prove  of  any  practical  value  in  the  diagnosis 
of  syphilis.  Finally,  the  work  of  Noguchi  and  Akatsu" 
proves  that  group  reactions  occur  between  certain  tre- 
ponemas and  that  specific  immune  bodies  capable  of  pro- 
ducing agglutination,  complement  fixation,  and  opsoniza- 
tion could  be  produced  in  the  blood  serum  of  rabbits  by 
injection  of  these  organisms.  They  worked  with  Tre- 
ponema pallidum,  Treponema  calligyriim,  Spirochceta  re- 
fringens,  Treponema  microdentium,  and  Treponema  mu- 
cosum,  and  found  that  the  pallidum  produced  higher  de- 
grees of  agglutination  and  complement  fixation,  than  the 
others,  but  that  group  reactions  with  complement  fixation 
occurred  between  Treponenia  pallidum  and  Treponema  cal- 
ligyrum,  between  calligyrum  and  Spirochceta  refringens,  and 
between  Treponema  microdentium  and  Treponema  muco- 
sum,  while  there  was  slight  group  reaction  between  the  lat- 
ter and  Treponema  pallidum. 

The  present  status  of  this  subject  is,  that  while  specific 
complement  fixation,  at  least  within  the  limits  of  a  group, 
is  possible  with  either  aqueous  or  alcoholic  extracts  of  pure 
cultures  of  Treponema  pallidum,  and  that  aqueous  extracts 


126  THE   WASSEEMANN   TEST 

give  the  best  results  there  is  no  evidence  sufficient  to 
prove  that  such  antigens  are  of  real  practical  value  in  the 
diagnosis  of  syphilis  by  the  Wassermann  test.  The  re- 
actions obtained  with  them  are  generally  weak  and  doubt- 
ful and  they  are  so  often  negative  where  the  ordinary  anti- 
genic extracts  are  positive  that  their  use  in  diagnosis  is 
not  justifiable.  Further  research  may  result  in  giving  us 
a  specific  antigen  for  the  AVassermann  test  but  at  present 
the  outlook  is  very  doubtful  that  such  an  antigen  can  be 
evolved. 


CHAPTER  VII 

THE  RESULTS  OF  THE  WASSERMANN  TEST  IN 
THE  VARIOUS  STAGES  OF  SYPHILIS.    TIME 
OF  APPEARANCE  OF  THE  REACTION. 
THE  SPECIFICITY  OF  THE  WAS- 
SERMANN TEST 

The  results  of  the  "VVassermann  test  vary  with  the  dif- 
ferent stages  of  syphilitic  infection,  the  highest  percentage 
of  positive  results  being  obtained  in  the  secondary  stage 
of  the  disease  and  the  lowest  in  latent  infections.  The 
results  also  vary  mth  the  methods  employed  by  ditferent 
observers,  although,  in  the  hands  of  experienced  serol- 
ogists,  the  results  obtained  with  one  method  are  practically 
the  same  as  those  with  another,  and  even  in  the  hands  of  the 
inexperienced  the  results  often  are  almost  as  good  as  when 
the  test  is  done  by  the  experienced  investigator.  It  is  one 
of  the  most  fortunate  thrags  connected  with  this  test,  that 
it  has  given  such  accurate  results  with  so  many  different 
modifications  of  the  original  test,  and  in  the  hands  of  such 
a  great  number  of  workers,  many  of  whom  were,  and  are, 
far  from  being  experts  in  serology.  As  Vedder  well  says :" 
''One  is  impressed  by  the  fact  that  the  Wassermann  re- 
action must  be  a  test  of  most  surprising  merit  to  have  sur- 
vived all  the  clumsy  technic  that  has  been  perpetrated  in 
its  name. ' ' 

The  figures  as  to  the  results  of  this  test  in  the  various 
stages  of  syphilis  which  are  here  given,  aside  from  those 
given  by  authorities  quoted,  are  based  upon  more  than 
fifty  thousand  Wassermann  tests  personally  made  by  the 
writer,  using  the  method  which  has  been  recommended  in 
Chapter  V.  A  great  many  thousands  of  these  tests  were 
made  upon  conditions  due  to  other  causes  than  syphilis 

127 


128  THE   WASSERMANN   TEST 

while  a  still  greater  number  were  reexaminations  made 
upon  the  same  case,  sometimes  as  many  as  twenty  or  more 
tests  having  been  made  upon  a  single  patient,  in  the  course 
of  months  or  years.  In  the  army  it  has  been  customary  to 
control  the  treatment  of  syphilis  by  repeated  Wassermann 
tests  and  this  practice  has  not  only  enabled  army  surgeons 
to  have  scientific  control  of  the  treatment  of  their  syphi- 
litic patients  but  has  also  enabled  us  to  have  a  very  ac- 
curate check  upon  the  efficiency  and  reliability  of  the  modi- 
fication of  the  Wassermann  test  which  is  recommended  in 
this  work.  The  writer  has  tried  to  keep  himself  informed 
as  to  its  success  in  the  hands  of  other  laboratory  workers 
and  it  is  gratifying  to  be  able  to  report  that  the  modification 
has  proved  satisfactory  in  every  laboratory  in  the  army 
in  which  it  has  been  adopted,  and  this  includes  nearly  all, 
and  that  it  has  been  recommended  as  a  standard  method 
for  all  army  laboratories  by  Lieut.  Col.  Vedder,^*  who  has 
devised  special  apparatus  which  makes  such  standardiza- 
tion possible. 

Before  considering  the  results  of  the  Wassermann  test 
in  each  stage  of  syphilis,  it  is  well  to  call  attention  to  the 
fact  that  many  of  the  statistics  which  are  to  be  found  in 
the  literature  are  based  upon  observations  made  during  the 
first  few  years  after  the  test  was  discovered  and  are,  there- 
fore, not  representative  of  the  results  that  are  really  being 
obtained  today.  A  great  amount  of  research  has  been  ex- 
pended upon  the  various  reagents  used  in  the  Wassermann 
test  and  a  great  manj^  factors  have  been  discovered  that 
influence  the  results  of  the  test,  in  one  way  or  another, 
so  that  at  the  present  time  much  better  results  are  being 
obtained  with  the  test  than  formerly  and  many  inaccura- 
cies in  technic  have  been  corrected.  This  fact  must  be 
borne  in  mind  in  considering  the  percentages  of  positive 
results  recorded  by  the  earlier  observers  in  the  various 
stages  of  syphilis  and  especially  the  numerous  reports  of 
positive  results  in  diseases  other  than  syphilis.  In  fact, 
the  results  now  being  obtained  with  this  test  are  hardly  at 


RESULTS   IN"   VARIOUS   STAGES   OF   SYPHILIS 


129 


all  comparable  with  those  which  were  obtained  with  it  dur- 
ing the  first  year  or  two  after  Wassermann's  publication 
of  his  technic. 

As  an  illustration  of  the  results  obtained  with  the  au- 
thor's method  in  the  various  stages  of  syphilis  Table 
XXVI  is  inserted,  giving  the  results  of  the  test  in  5,600 
syphilitic  infections,  personally  tested  by  him: 

Table  XXVI 
The  Results  of  the  Wassermann  Test  in  5,600  Cases  of  Syphilis 


STAGE 

total  cases 

positive 

% 

negative 

% 

Primary 

1080 

970 

89.8 

110 

10.1 

Secondary 

2217 

2132 

96.1 

85 

4.9 

Tertiary 

728 

633 

87.4 

95 

13.5 

Latent 

1525 

1039 

68.1 

486 

31.8 

Congenital 

28 

25 

82.2 

3 

10.7 

Parasyphilis 

22 

15 

68.1 

7 

31.8 

totals 

5600 

4S14 

85.9 

786 

14 

In  the  above  cases  no  test  was  considered  positive  un- 
less there  was  complete  inhibition  of  hemolysis  (a  four- 
plus  reaction)  except  in  the  very  early  primary  cases  when 
a  three-plus  reaction  was  considered  positive  in  a  few  in- 
stances. By  insisting  upon  absolution  inhibition  of  hem- 
olysis as  indicating  a  positive  reaction  in  these  cases  it  is 
undoubtedly  true  that  some  cases  of  infection,  which  gave 
reactions  almost  absolute,  have  been  erroneously  consid- 
ered as  negative,  but  in  most  of  the  army  laboratories  we 
have  not  felt  justified  in  basing  a  positive  diagnosis  of 
syphilis  upon  anything  less  than  absolute  inhibition  of 
hemolysis.  In  considering  this  table  it  should  also  be  re- 
membered that  in  at  least  95  per  cent  of  the  cases  tabulated 
only  one  Wassermann  test  was  performed  and  that  had 
repeated  tests  been  made  it  is  undoubtedly  true  that  a 
higher  percentage  of  positive  results  would  have  been  ob- 
tained in  all  of  the  stages  of  syphilis  tested. 

Our  results  are  in  practical  agreement  with  those  of 
most  recent  observers,  so  that  it  may  be  stated  that  about 
86  per  cent  of  infections  with  syphilis  will  give  a  positive 


130  THE   WASSERMANN   TEST 

reaction  with  the  Wassermann  test,  if  they  are  distributed 
among  the  various  stages  as  in  the  table  given ;  that  10 
per  cent  of  primary  cases  will  give  a  negative  result ;  about 
5  per  cent  of  secondary  cases;  about  13  per  cent  in  the 
tertiary  stage;  and  about  30  per  cent  in  the  latent  stage 
of  the  disease.  With  good  technic  and  efficient  antigenic 
extracts  one  should  obtain  90  per  cent  of  positive  results  in 
the  primary  stage  of  syphilis;  95  per  cent  of  positive  re- 
sults in  the  secondary  stage;  87  per  cent  of  positive  results 
in  the  tertiary  stage;  and  70  per  cent  of  positive  results 
in  the  latent  stages. 

Results  in  the  Primary  Stage  of  Syphilis. — It  is  now  a 
generally  accepted  fact  that  the  earlier  a  syphilitic  infec- 
tion is  diagnosed  the  better  will  be  the  results  of  any 
method  of  treatment  that  may  be  instituted.  Thus  the 
early  diagnosis  of  the  disease  becomes  of  paramount  im- 
portance and  with  the  dark-field  apparatus  and  the  Was- 
sermann  test  Ave  are  supplied  with  agencies  that,  if  prop- 
erly used,  should  enable  us  to  make  the  diagnosis  during 
the  early  portion  of  the  primary  stage  of  the  disease  in 
the  vast  majority  of  infections. 

If  possible  every  patient  exhibiting  a  sore  upon  the 
penis  should  be  given  the  benefit  of  an  examination  of  the 
serum  from  the  sore  by  the  dark-field  apparatus.  If  this 
were  done,  the  Treponema  pallidum  would  be  found  in 
most  instances  of  true  syphilitic  infection,  and  thus  the  dis- 
ease would  be  diagnosed  at  a  very  early  stage.  However, 
the  dark-field  apparatus  is  often  unobtainable  and  the  ex- 
amination can  not  be  made,  so  that  the  question  arises  as  to 
the  diagnostic  value  of  the  Wassermann  test  in  the  primary 
stage  of  syphilis.  If  one  were  to  believe  some  writers  it 
would  be  concluded  that  during  this  stage  of  syphilis  the 
Wassermann  lest  is  of  very  little  value,  as  so  many  cases 
give  a  negative  reaction,  but  this  has  not  been  the  writer's 
experience  with  his  modification  of  the  test,  for,  as  already 
mentioned,  practically  90  per  cent  of  the  primary  cases 
he  has  tested  have  given  a  positive  result  before  the  de- 


RESULTS   IN   VARIOUS   STAGES   OF   SYPHILIS  131 

velopment  of  secondary  symptoms.  Therefore,  if  a  dark- 
field  examination  can  not  be  made,  or  is  negative,  it  is 
recommended  that  every  patient  presenting  a  suspicious 
lesion  upon  the  penis  be  given  a  AVassermann  test,  for  if 
modern  research  upon  syphilis  has  proved  anything,  it  is 
that  the  morphology  of  a  sore  upon  the  penis  is  of  much 
less  value  than  formerly  supposed,  and  that  the  typical 
Hunterian  chancre  is  almost  as  much  the  exception  as  the 
rule.  Mixed  infections  of  chancre  and  chancroid,  with  the 
resultant  confusion  in  clinical  pictures,  have  been  quite 
common  in  the  writer's  experience,  and  with  the  admis- 
sions by  the  best  authorities  upon  syphilis  that  it  is  often 
impossible  to  diagnose  syphilis  from  the  appearance  of  the 
initial  lesion,  the  use  of  the  Wassermann  test  and  the  dark- 
field  apparatus  in  every  suspicious  case  is  imperative. 

From  experience  and  experimental  observations  it  is 
certain  that  a  syphilitic  infection  must  exist  for  a  certain 
length  of  time  in  man  before  the  substance  or  substances 
which  give  rise  to  a  positive  AVassermann  reaction  appear 
in  the  blood.  Just  how  long  this  is  will  vary  with  the  viru- 
lence of  the  infection  and  the  resisting  power  of  the  pa- 
tient, but  it  is  beyond  controversy  that  sometimes  the  test 
proves  positive  a  very  few  days  after  the  appearance  of  the 
initial  lesion,  although  this  occurs  but  rarely.  The  writer 
has  observed  and  reported'^^  one  case  in  which  the  reaction 
occurred  only  five  days  after  the  appearance  of  the  chancre 
and  two  cases  in  which  it  occurred  after  eight  days  of  the 
appearance  of  the  initial  lesion.  As  will  appear  in  Table 
XXIX,  no  less  than  27  of  77  cases,  or  36  per  cent  of 
primary  cases  tested  by  the  writer,  gave  a  positive  reaction 
during  the  first  week  after  the  chancre  appeared  and  al- 
most 60  per  cent  during  the  second  week  after  the  appear- 
ance of  the  chancre.  It  is  thus  evident,  that  even  very 
early  in  the  primary  stage  of  syphilis  a  positive  Wasser- 
mann reaction  may  occur,  and  thus  the  test  will  prove  of 
value  in  diagnosis. 

As  would  be  expected,  the  statistics  given  by  different  au- 


132 


THE   WASSERMANN   TEST 


thorities  regarding  the  percentage  of  positive  results  in 
the  primary  stage  of  syphilis  vary  greatly,  especially  those 
published  shortly  after  Wassermann's  original  paper  upon 
the  subject.  Kolmer^®  states  that  in  general,  in  primary 
syphilis  positive  results  will  be  found  in  from  80  to  90 
per  cent  of  the  cases  tested,  and  the  writer  agrees  with 
this  statement,  as  it  has  been  his  experience  that  when 
properly  performed  th-e  Wassermann  test  should  give  at 
least  this  number  of  positive  results.  Table  XXVII  gives 
the  results  obtained  by  different  observers  in  the  primarj^ 
stage  of  syphilis : 

Table  XXVII 

Eesults  of  the  Complement-fixation  Test  for  Syphilis  in  the  Primary 
Stage  of  the  Disease 


NAME   OF    observer 

NO.  OP  CASES 

POSITIVE 

%   POSITIVE 

Arning 
Bering 
Boas 

48 

56 

50 

25 

47 
30 

60 
84 
60 

Bruek-Stern 

27 

13 

48.5 

Fischer-Meier 

8 

6 

75 

Grosser 

20 

19 

95 

Hancken 

17 

15 

88 

Craig 
Kaplan 
Noguchi 
Swift 

1080 

138 

70 

16 

970 

125 

65 

13 

89.8 

90 

92.8 

81 

Vedder 

242 

178 

73.5 

From  this  table  it  is  evident  that  the  percentage  of  posi- 
tive results  with  the  Wassermann  test  in  the  primary  stage 
of  syphilis  has  varied  greatly  in  the  hands  of  different  ob- 
servers, the  percentage  being  as  low  as  48  per  cent  and  as 
high  as  95  per  cent.  However,  as  already  stated,  from  80 
to  90  per  cent  of  the  usual  run  of  primary  infections  com- 
ing to  the  physician  should  give  a  positive  reaction.  Of 
course,  if  tests  were  made  upon  a  very  large  number  of 
very  early  infections  in  a  series  reported,  the  percentage 
would  be  much  lower  than  if  cases  were  tested  from  two 
to  three  weeks  after  the  appearance  of  the  initial  lesion, 
but  the  writer's  experience  has  been  that  at  least  80  per 
cent  of  cases  of  primary  syphilis  should  give  a  positive 


RESULTS   IF   VARIOUS   STAGES   OF   SYPHILIS  133 

Wassermaim  test  and  it  is  believed  that,  in  the  army,  the 
initial  lesion  is  reported  to  the  surgeon  at  least  as  soon 
as  it  is,  in  the  vast  majority  of  cases,  in  civil  life. 

It  is  true  that  the  percentage  of  positive  results  with  this 
test  varies  more  in  the  primary  stage  of  syphilis  than  in 
other  stages,  with  the  possible  exception  of  the  latent  stage, 
due  to  many  reasons,  among  the  most  important  being 
that  the  test  is  made  within  a  very  few  days  of  the  ap- 
pearance of  the  initial  lesion  or  early  treatment  often  ob- 
scures the  reaction  at  this  stage  of  the  disease.  The 
writer  has  often  observed  negative  cases  become  positive 
with  the  cessation  of  treatment,  during  the  primary  stage, 
and  hundreds  of  instances  in  which,  in  untreated  cases, 
a  negative  reaction  became  positive  after  an  interval  of 
days  or  weeks,  and  before  the  occurrence  of  secondary 
symptoms.  It  should  be  remembered,  however,  that  a  cer- 
tain proportion  of  negative  results  must  be  expected  in  the 
primary  stage  of  syphilis  and  that  a  negative  result  is  no 
proof  that  a  suspicious  lesion  is  not  syphilitic. 

Time  of  Appearance  of  the  Reaction 

The  date  after  infection  with  syphilis  that  one  may  ex- 
pect the  Wassermann  reaction  to  be  positive  is  a  point  of 
great  practical  interest,  for  the  earlier  treatment  is  com- 
menced in  syphilis  the  better  are  the  therapeutic  results, 
and  unless  the  Wassermann  test  can  assist  in  diagnosis 
early  in  the  disease,  it  loses  much  of  its  value  as  a  diag- 
nostic agent.  While  the  ideal  method  of  diagnosing  syph- 
ilis in  the  primary  stage  is  by  the  demonstration  of  Tre- 
ponema pallidum  with  the  dark-field  apparatus,  this  meas- 
ure is  not  infrequently  unsuccessful,  especially  if  local 
treatment  has  been  given  the  initial  lesion,  and,  in  addi- 
tion, comparatively  few  practitioners  possess  the  appara- 
tus. The  demonstration  of  the  treponema  in  stained  prep- 
arations or  by  the  India  ink  method  should  always  be  at- 
tempted but  these  methods  are  also  often  unsuccessful,  so 


134 


THE   WASSERMANN   TEST 


that  one  must  depend  upon  the  Wassermann  test  fre- 
quently for  an  early  diagnosis. 

Contrary  to  the  opinion  of  some  observers,  the  writer 
believes  that  this  test  is  a  most  valuable  aid  to  the  diag- 
nosis of  syphilis  even  thus  early  in  the  disease,  for,  in  his 
experience,  nearly  90  per  cent  of  primary  cases  tested  by 
him  have  shown  a  positive  result  before  the  appearance  of 
secondary  symptoms,  and  while  a  single  test  performed  at 
a  very  early  date  after  the  appearance  of  the  chancre  will 
be  negative  in  a  very  large  proportion  of  cases,  repeated 
tests  will  prove  that  the  reaction  becomes  positive  before 
the  appearance  of  secondary  symptoms  in  most  patients. 

In  Table  XXVIII  Vedder"  shows  the  results  of  the  ex- 
amination of  cases  of  syphilis  in  the  primary  stage  by  both 
the  dark-field  apparatus  and  the  Wassermann  test,  and  it 
is  of  interest  as  demonstrating  that  both  methods  fail,  at 
times,  to  aid  in  the  diagnosis  of  the  disease. 

Table  XXVIII 

Result  op  Examination  of  Primary  Cases  of  Syphilis  by  Dark-Field  and 
THE  Wassermann  Test* 


duration  of 

dark- 

WASSER- 

DURATION OF 

DARK- 

WASSER- 

SORE 

field 

MANN 

SORE 

FIELD 

MANN 

2  days 

-1- 

_ 

1  month 

_ 

+  + 

3  days 

-f 

- 

7  days 

+ 

4--^ 

7  days 

+ 

_ 

9  days 

+ 

+ 

7  days 

+ 

- 

14  days 

+ 

+ 

10  days 

+ 

_ 

15  days 

-1- 

+ 

2  weeks 

+ 

_ 

1  month 

+ 

+  + 

2  weeks 

+ 

_ 

1  month 

+ 

+ 

3  weeks 

+ 

+  - 

1  month 

+ 

+  4- 

3  weeks 

+ 

_ 

1  month 

+ 

+  + 

3  weeks 

+ 

_ 

1  month 

+ 

+  -I- 

1  month 

+ 

_ 

1  month 

+ 

+  + 

1  week 

_ 

+  + 

11/^  months 

+ 

+  + 

1  week 

_ 

+ 

2  months 

+ 

+  + 

2  weeks 

_ 

+  + 

1  month 

_ 

_ 

3  weeks 

_ 

+ 

1  month 

_ 

_ 

1  month 

- 

+  4- 

*The  sign  +  +  equals  absolute  inhibition  of  hemolysis,  the  four-plus  reaction  of  many 
laboratories. 


Table  XXVIII  is  conclusive  evidence  of  the  value  of  the 
Wassermann  test  as  an  aid  in  the  diagnosis  of  syphilis  in 


KESULTS   IN   VARIOUS   STAGES   OF   SYPHILIS 


135 


the  primary  stage  of  syphilis.  It  will  be  noted  that  in  no 
less  than  three  cases  the  Wassermann  test  was  positive 
one  week  after  the  appearance  of  the  chancre  and  that  in 
two  of  these  cases  the  dark-field  examination  Avas  negative. 
The  table  also  demonstrates  that  in  every  case  of  primary 
syphilis  both  a  dark-field  examination  and  a  Wassermann 
test  should  be  made,  if  possible. 

Table  XXIX,  compiled  from  personal  tests,  gives  the 
date  of  appearance  of  the  positive  reaction  in  periods  of 
weeks  after  the  appearance  of  the  initial  lesion.  Six  hun- 
dred cases  of  primary  syphilis  are  here  considered  and  the 
Wassermann  tests  were  performed  by  the  writer  in  the 
ordinary  course  of  routine  examinations  in  the  army.  A 
great  many  more  cases  of  primary  infection  were  really 
tested  but  are  not  included  for  the  reason  that  we  could 
not  be  sure  of  the  data  regarding  the  date  of  appearance  of 
the  initial  lesion. 

Table  XXIX 

Date  of  Appearance  of  Wassermann  Eeaction  in  Weeks  in  600  Cases  of 

Primary  Syphilis 


WEEK  AFTER 

appearance 

TOTAL    cases 

POSITIVE 

% 

NEGATIVE 

% 

of  chancre 

First  week 

77 

27 

36.3 

50 

64.9 

Second  week 

155 

92 

59.3 

63 

40.3 

Third  week 

158 

109 

68.9 

49 

31. 

Fourth  week 

167 

129 

77.2 

38 

22.7 

Fifth    week 

43 

35 

81.3 

8 

18.6 

From  this  table  it  is  evident  that,  in  the  writer's  ex- 
perience, 36  per  cent  of  cases  of  primary  syphilis  have 
given  a  positive  Wassermann  reaction  by  the  end  of  the 
first  week  after  the  appearance  of  the  initial  lesion ;  almost 
60  per  cent  by  the  end  of  the  second  week;  almost  70  per 
cent  by  the  end  of  the  third  week ;  over  77  per  cent  by  the 
end  of  the  fourth  week ;  and  over  80  per  cent  by  the  end  of 
the  fifth  week  after  the  appearance  of  the  chancre. 

It  is  also  evident  that  this  test  is  of  very  distinct  value 
in  the  diagnosis  of  syphilis  during  the  primary  stage  of 


136  THE   WASSERMANIT   TEST 

the  disease,  and  should  always  be  resorted  to  if  a  dark- 
field  examination  is  impossible  or  gives  a  negative  result. 
A  single  negative  test,  at  this  stage  especially,  should  not 
be  considered  as  proving  that  a  suspected  lesion  is  non- 
syphilitic,  but  repeated  tests  should  be  made  before  a  pa- 
tient is  assured  that  the  condition  present  is  not  due  to 
syphilis.  In  the  armj^,  owing  to  the  situation  of  the  va- 
rious Department  Laboratories,  the  Wassermann  test  is  al- 
ways available  and  suspected  cases  can  be  examined  at 
weekty  intervals,  a  practice  which  enables  the  army  sur- 
geon to  establish  the  diagnosis  very  early  in  the  disease, 
but  in  civil  practice  this  is  generally  impossible,  omng  to 
the  cost  of  the  test.  However,  whenever  possible,  repeated 
Wassermann  tests  should  be  made  upon  patients  suspected 
of  presenting  the  primary  lesion  of  syphilis. 

As  regards  the  question  as  to  how  early  the  positive 
Wassermami  may  appear  in  the  blood  after  the  primary 
lesion  appears,  there  are  a  considerable  number  of  obser- 
vations upon  record  showing  that  positive  reactions  have 
been  obtained  as  early  as  the  third  and  fourth  days  after 
the  appearance  of  the  chancre,  and  a  few  in  which  a  posi- 
tive reaction  has  been  reported  even  before  the  appearance 
of  the  initial  lesion.  In  view  of  our  knowledge  of  the  na- 
ture of  the  syphilitic  poison  it  may  be  stated  that  one  must 
regard  with  suspicion  the  reports  of  a  positive  "Wasser- 
mann before  a  primary  lesion  was  observed,  for  it  is  prac- 
tically impossible  to  rule  out  previous  syphilitic  disease 
in  most  patients  and  unless  this  can  be  done  or  it  can  be 
definitely  proved  that  the  lesion  supposed  to  be  primary 
in  such  cases  is  really  so,  by  the  demonstration  of  Tre- 
ponema 'pallidum,  the  evidence  can  not  be  considered  as  con- 
clusive. 

However,  it  may  be  accepted  as  proved  beyond  a  doubt 
that  the  Wassermann  test  is  not  so  very  infrequently  posi- 
tive by  the  end  of  the  first  Aveek  after  the  appearance  of 
the  chancre,  and  the  writer  has  observed  two  cases  in  which 
it  was  positive  five  days  after  the  sore  was  first  noticed, 


RESULTS   IN   VARIOUS   STAGES   OF    SYPHILIS  137 

and  in  both  cases  Treponema  pallidum  was  demonstrated 
in  serum  from  the  lesions.  Well  authenticated  cases  are 
on  record  in  which  the  test  became  positive  two  or  three 
days  after  the  initial  lesion  was  noted,  so  that  the  possi- 
bility of  a  positive  Wassermann  test  before  the  appearance 
of  such  a  lesion  can  not  be  denied,  although  it  must  be  ex- 
ceedingly rare. 

It  is  now  generally  conceded  that  when  the  primary  le- 
sion of  syphilis  appears  the  disease  has  already  become  a 
generalized  infection,  so  that  it  is  not  difficult  to  under- 
stand why  a  positive  Wassermann  reaction  may  occur  very 
soon  after  the  chancre  appears,  and  the  variation  that  oc- 
curs as  to  the  time  of  the  appearance  of  the  positive  re- 
action in  different  patients  must  depend  upon  the  virulence 
of  the  treponema  and  the  reaction  of  the  tissues  of  the 
patient  to  the  organism.  The  writer  has  observed  many 
cases  in  which  repeated  Wassermann  tests  always  proved 
negative,  though  there  was  no  question  of  the  presence  of 
syphilis  and  marked  lesions  were  present  at  the  time  that 
the  tests  were  made,  so  that  in  a  certain  percentage  of 
cases  of  syphilis  it  is  probable  that  a  positive  reaction  will 
never  be  obtained.  The  writer  has  also  observed  tAvo  pa- 
tients in  whom  the  primary  lesion  of  syphilis  was  present, 
as  demonstrated  by  the  finding  of  Treponema  pallidum  by 
the  dark-field,  and  whose  blood  serum  gave  a  positive  re- 
action prior  to  the  development  of  secondary  symptoms,  but 
in  whom  no  secondary  symptoms  ever  developed,  although 
they  were  under  observation  for  nearly  one  year,  and  in 
whom  the  Wassermann  reaction  became  negative,  although 
no  specific  treatment  was  ever  administered.  In  his  opin- 
ion, these  cases  demonstrate  that  syphilis  is  sometimes 
spontaneously  cured,  and  it  is  not  improbable  that  this 
occurs  much  more  frequently  than  is  generally  supposed. 

In  view  of  the  reports  of  positive  Wassermann  reactions 
occurring  before  the  initial  lesion  appears,  the  recent  work 
of  Woods  and  Morris^^  upon  complement  fixation  in 
trypanosomiasis  is  very  suggestive.    They  found  that  with 


138  THE   WASSEKMANN   TEST 

an  antigen  prepared  from  the  spleen  of  a  rat  heavily  in- 
fected with  Trypanosoma  equiperdum,  complement  fixa- 
tion usually  appeared  in  an  infected  animal's  blood  serum 
within  eight  days  after  inoculation,  usually  at  the  time 
that  the  trypanosomes  appeared  in  the  blood  of  the  infected 
animal,  but  sometimes  even  before  this  occurred.  In  every 
instance,  however,  complement  fixation  preceded  the  ap- 
pearance of  symptoms  of  the  infection. 

Results  in  the  Secondary  Stage  of  Syphilis. — It  is  in  the 
secondary  stage  of  syphilis  that  the  highest  percentage  of 
positive  results  are  obtained  with  the  Wassermann  re- 
action. The  per  cent  of  positive  reactions  reported  by  va- 
rious observers  varies  somewhat,  but  it  may  be  stated  that 
in  this  stage  of  the  disease  practically  95  per  cent  of  the 
patients  should  give  a  positive  result,  provided,  of  course, 
that  treatment  has  not  been  administered,  but  even  in  such 
cases  one  would  expect  from  90  to  95  per  cent  of  positive 
results.  Kolmer^^  states  that  in  untreated  secondary  syph- 
ilis he  has  never  obtained  a  negative  result  and  Boas^°  in 
437  untreated  secondary  cases  obtained  100  per  cent  of 
positive  reactions.  The  writer,  in  2,217  cases  of  secondary 
syphilis,  many  of  whom  had  received  some  specific  treat- 
ment, obtained  2,132  positive  results,  or  96.1  per  cent,  and 
Vedder,®^  in  310  secondary  cases  of  similar  nature,  obtained 
91  per  cent  of  positive  results. 

Table  XXX  gives  the  results  obtained  in  the  secondary 
stage  of  the  disease  by  various  observers : 

From  this  table  it  will  be  noted  that  the  percentage  of 
positive  results  in  the  secondary  stage  of  syphilis  varies, 
according  to  different  observers,  from  79  per  cent  to  100 
per  cent,  but  it  should  be  remembered  that  many  of  the 
cases  tested  by  one  observer  ma^^  have  had  specific  treat- 
ment, while  those  tested  by  another  had  had  no  treatment 
whatever,  so  that  the  low  percentages  reported  can  not  be 
considered  as  giving  any  indication  of  the  real  value  of  the 
test  in  the  absence  of  data  regarding  specific  treatment. 


RESULTS  IN  VARIOUS  STAGES  OF  SYPHILIS 


139 


Table  XXX 

The  Results  of  the  Complement-fixation  Test  for  Syphilis  in  the 
Secondary  Stage  of  the  Disease 


observer 

NO.  of  cases 

positive 

%  positive 

Arning 

107 

99 

93 

Bering 

113 

111 

98 

Druck- Stern 

115 

101 

87.1 

Blumenthal-Roscher 

131 

130 

99 

Craig 

2217 

2132 

96.1 

Hoene 

376 

260 

79.1 

Lesser 

204 

186 

91 

Lederman 

110 

108 

98 

Merz 

377 

366 

97 

Noguchi 

197 

190 

.       96 

Schonnefeld 

112 

112 

100 

Swift 

76 

70 

92 

Vedder 

310 

285 

91.9 

As  already  stated,  the  percentage  of  positive  results  in 
untreated  cases  of  secondary  syphilis  should  be  something 
over  95  per  cent,  and  unless  as  high  a  percentage  as  this 
is  obtained  the  complement-fixation  system  used  should  be 
carefully  studied  for  the  occurrence  of  possible  errors,  as 
the  Wassermann  test,  as  at  present  performed,  should  not 
be  given  a  lower  percentage  of  positive  results  in  the 
secondary  stage  of  syphilis  than  that  mentioned. 

A  negative  reaction  in  this  stage  of  syphilis,  when  there 
is  a  clear  history  of  infection  and  lesions  are  present,  is 
of  no  value  whatever  in  excluding  the  disease ;  and  as  nega- 
tive reactions  may  occur,  even  when  the  test  is  repeated 
many  times  during  this  stage  of  the  disease,  the  diagnosis 
of  syphilis  should  be  made,  in  such  instances,  without  ref- 
erence to  the  result  of  the  test.  It  should  also  be  remem- 
bered that  the  severity  of  the  lesions  present  has  nothing 
to  do  with  the  type  of  reaction  obtained,  for  cases  with 
very  mild  symptoms  will  frequently  react  as  strongly  as 
those  showing  severe  lesions,  and  not  infrequently  a  pa- 
tient presenting  the  most  pronounced  secondary  eruption 
and  mucous  patches  will  give  a  weak  Wassermann  reaction, 
which,  unless  repeated,  might  be  interpreted  as  doubtful. 
However,  in  these  cases  re]3eated  Wassermann  tests  almost 


140 


THE   WASSERMANN   TEST 


invariably  result  in  the  appearance  of  a  strong  positive  re- 
action. 

Results  in  the  Tertiary  Stage  of  the  Disease. — ^Perhaps 
in  no  other  stage  of  syphilis  has  the  Wassermann  test 
proved  as  valuable  as  in  the  tertiary  stage,  and  especially 
in  those  cases  in  which  there  is  involvement  of  the  central 
nervous  system.  The  percentage  of  positive  results  ob- 
tained will  vary,  of  course,  with  the  amount  of  treatment 
that  has  been  given  the  patient,  for  in  a  large  majority 
of  the  cases  of  tertiary  syphilis,  observed  in  practice,  a 
considerable  amount  of  specific  treatment  has  been  admin- 
istered prior  to  the  time  that  the  blood  was  collected  for 
a  Wassermann  test.  If  cases  are  tested  that  have  received 
no  specific  treatment  and  in  which  active  tertiary  lesions 
are  present,  the  percentage  of  positive  results  should  be 
above  95  per  cent,  but  in  the  usual  run  of  cases  examined 
the  Wassermann  test,  if  properly  applied,  should  give  posi- 
tive results  in  from  80  to  85  per  cent  of  cases  examined. 
The  writer,  in  728  cases  of  tertiary  syphilis  obtained  633 
positive  results,  or  87.4  per  cent,  using  the  method  recom- 
mended in  this  work,  and  Vedder,®^  using  the  same  method, 
obtained  227  positive  reactions  in  263  cases  tested,  or  86.3 
per  cent. 

Table  XXXI  gives  the  results  reported  by  various  ob- 


Table  XXXI 

Results  of  the  Complement-fixation  Test  for  Syphilis  in  the  Tertiary 
Stage  of  the  Disease 


observer 

no.  of  cases  tested 

positive 

%  positive 

Arning 

30 

27 

90 

Bering 

45 

37 

82 

Bruek-Stern 

47 

27 

57 

Fleishman 

41 

40 

98 

Hohne 

33 

21 

63 

Craig 

728 

633 

87.4 

Lederman 

78 

75 

96 

Lesser 

131 

119 

90 

Merz 

158 

127 

80.3 

Noguchi 

177 

159 

89.9 

Swift 

45 

37 

80 

Vedder 

263 

227 

86.3 

KESULTS   1^   VARIOUS   STAGES   OF   SYPHILIS  141 

servers  of  the  complement-fixation  test  for  syphilis  dur- 
ing the  tertiary  stage  of  the  disease. 

From  this  table  it  is  evident  that  the  results  of  the  Was- 
sermann  test  in  the  tertiary  stage  of  syphilis  varied  from 
57  per  cent  to  96  per  cent,  according  to  various  observers. 
This  great  variation  in  the  results  may  probably  be  ex- 
plained by  certain  of  the  observers  including  under  the 
tertiary  stage  cases  tested,  1,  cases  without  definite  symp- 
toms; 2,  cases  that  had  received  specific  treatment  within 
a  recent  date  or  were  receiving  treatment;  and  3,  cases  in 
which  tertiary  symptoms  had  never  appeared.  It  is  evi- 
dent that,  until  statistics  are  based  upon  similar  cases  a 
great  deal  of  variation  must  be  expected  in  the  results  of 
this  test  in  the  hands  of  different  observers. 

In  syphilitic  disease  of  the  blood  vessels,  as  aortitis, 
aortic  aneurism,  and  aortic  insufficiency,  the  Wassermann 
test  has  proved  of  the  very  greatest  diagnostic  value  and 
in  syphilitic  disease  of  the  central  nervous  system  the  test 
is  invaluable.  This  subject  will  be  discussed  more  fully 
in  Chapter  X. 

Results  in  the  Latent  Stages  of  the  Disease. — The  Was- 
sermann test  in  the  latent  stages  of  syphilis  has  been  of 
the  greatest  use  in  indicating  that  treatment  was  neces- 
sary in  patients  thought  to  be  cured  of  the  infection  be- 
cause of  the  absence  of  symptoms  for  long  periods  of  time. 
There  is  probably  no  disease  occurring  commonly  in  the 
practice  of  the  physician  which  so  often  exhibits  intervals 
during  which  clinical  symptoms  are  absent  as  does  syph- 
ilitic infection  and  often  a  positive  Wassermann  reaction 
is  the  only  tangible  evidence  of  the  disease.  The  patho- 
logical investigations  of  careful  workers,  as  Warthin,  have 
shown  that  the  human  body  may  be  extensively  invaded  by 
Treponema  pallidum  without  clinical  symptoms  being  evi- 
dent and  with  a  positive  Wassermann  reaction  as  the  only 
evidence  of  such  infection,  and  one  of  the  most  valuable  of 
assets  of  the  Wassermann  test  is  its  capacity  for  detecting 
such  infections.    Of  course,  the  test  is  frequently  negative 


142 


THE   WASSERMANN   TEST 


/in  this  class  of  patients,  as  would  be  expected,  for,  as  the 
writer  has  shown,  a  certain  amonnt  of  the  substance  or 
substances  causing  complement  fixation  in  syphilis  must 
be  present  in  order  to  accomplish  this  result,  and  in  the 
latent  cases  of  syphilis  a  sufficient  amount  is  not  present 
in  a  considerable  percentage  of  cases,  as  shown  by  the  re- 
sults of  the  test. 

The  writer  has  tested  1,525  cases  of  latent  syphilis,  with 
a  positive  reaction  in  1,039,  or  68  per  cent,  using  the  method 
of  performing  the  test  recommended  in  this  work.  Ved- 
der,^^  using  the  same  method,  tested  114  cases  with  a  posi- 
tive result  in  92,  or  80.7  per  cent.  Boas,^*  in  363  cases  of 
early  latent  syphilis  obtained  positive  results  in  only  40 
per  cent  of  the  cases,  Avhile  in  older  cases  he  obtained  posi- 
tive results  in  but  22  per  cent  of  treated  cases  but  in  cases 
treated  carelessly  the  positive  results  equaled  74  per  cent. 

Table  XXXII  gives  the  results  of  the  Wassermann  test 
obtained  by  the  observers  mentioned  in  the  latent  stage  of 
syphilitic  infection. 

Table  XXXII 

Eesult  of  the  Complement-fixation  Test  for  Syphilis  in  the  Latent 
Stages  of  the  Disease 


OBSERVER, 

NO.  OF  CASES  TESTED 

POSITIVE 

%    POSITIVE 

Bering 

147 

75 

48 

Grosser 

35 

12 

33.3 

Craig 

1525 

1039 

68.1 

Fox 

54 

25 

46 

Lederman 

78 

36 

46 

Noguchi 

265 

206 

77.7 

Swift 

39 

25 

64 

Vedder 

114 

92 

80.7 

As  would  be  expected,  there  is  a  great  variation  in  the 
number  of  positive  reactions  obtained  with  the  test  in  the 
latent  stages  of  syphilis,  as  shown  in  this  table.  When  it 
is  remembered  that  in  most  of  the  cases  tested  specific 
treatment  had  been  given  for  a  longer  or  shorter  period 
of  time ;  that  there  were  no  symptoms  of  the  disease  pres- 
ent at  the  time  the  blood  was  tested :  and  that  in  some  the 


RESULTS    IN"    VARIOUS    STAGES    OF    SYPHILIS  143 

only  history  of  a  syphilitic  infection  was  a  doubtful  one; 
the  low  percentage  of  positive  results  sometimes  obtained 
in  this  class  of  cases  can  be  understood.  In  the  writer's 
cases  there  was  always  a  clear  history  of  infection  but  in 
the  majority  of  the  patients  some  specific  treatment  had 
been  taken,  so  that  the  percentage  of  positive  results  ob- 
tained ;  i.  e.,  68  per  cent,  may  be  considered  the  average 
percentage  obtained  in  latent  infections  in  such  cases. 

The  value  of  the  "Wassermann  test  in  the  diagnosis  of 
syphilis  in  the  latent  periods  of  the  disease  can  not  be 
overestimated  either  from  the  sociological  or  therapeutic 
standpoint.  The  sociological  aspect  of  latent  syphilitic  in- 
fection will  be  treated  of  more  fully  in  the  discussion  of  the 
subject  of  Wassermann  surveys,  but  it  can  not  be  too 
strongly  emphasized  that  there  is  a  very  large  amount  of 
sj'-philitic  infection  present  in  all  countries  that  is  never 
recognized  and  it  has  only  been  within  very  recent  years 
that,  because  of  researches  upon  various  classes  of  the 
population  by  means  of  the  Wassermann  test,  it  has  been 
realized  to  what  a  great  extent  this  insidious  disease  has 
invaded  society. 

From  the  therapeutic  standpoint,  the  Wassermann  test 
is  most  valuable  in  the  latent  stage  of  syphilitic  infection 
because  a  positive  reaction  is  the  onl^^  index  which  we  pos- 
sess as  to  the  existence  of  the  infection  and  the  necessity  for 
specific  treatment.  The  test  is  often  positive  even  after 
specific  treatment  has  been  administered  for  months  and 
years,  and  the  vast  majority  of  patients  treated  with  mer- 
cury, before  the  days  of  salvarsan,  and  supposed  to  have 
been  cured,  will  give  a  positive  reaction  with  the  Wasser- 
mann test.  Some  authorities  have  gone  so  far  as  to  state 
that  they  do  not  believe  that  any  syphilitic  infection  was 
ever  cured  by  mercury  alone,  because  of  the  revelations  of 
the  Wassermann  test  in  latent  infections,  but  the  writer 
believes  this  to  be  too  strong  a  statement,  although  it  must 
be  admitted  that  the  cases  proving  the  contrary  are  com- 
paratively few  in  number. 


144 


THE   WASSERMANN   TEST 


Results  in  Congenital  Syphilis. — The  results  of  the  Was- 
sermann  test  in  congenital  syphilis  vary  with  the  character 
of  the  case  tested.  In  children  showing  the  lesions  of 
syphilis  at  birth  the  test  gives  100  per  cent  of  positive 
results,  in  the  writer's  experience,  but  in  children  exhib- 
iting the  lesions  of  late  congenital  syphilis  the  percentage 
of  positive  results  is  lower,  averaging  from  80  to  85  per 
cent.  In  still  older  individuals  in  whom  the  disease  is 
supposed  to  be  congenital  the  percentage  of  positive  re- 
sults will  not  exceed  70  to  75  per  cent. 

Table  XXXIII  gives  the  results  obtained  by  several  ob- 
servers with  the  Wassermann  test  in  congenital  syphilis: 

Table  XXXIII 

Results  of  the  Complement-fixation  Test  for  Syphilis  in  Congenital 

Syphilis 


observer 

NO.  OF  cases  tested 

positive 

%  positive 

Arning 

5 

3 

60 

Craig 

28 

25 

82.2 

Jesionek-Meirosky 

18 

16 

88.8 

Noguchi 

17 

17 

100 

Vedder 

128 

123 

96 

The  variation  obtained  by  different  observers  in  the  re- 
sults of  the  Wassermann  test  in  congenital  syphilis  are 
well  shown  in  the  table  and  depend  upon  the  causes  already 
mentioned.  The  recent  work  of  Veeder^^  upon  the  subject 
is  of  interest.  He  studied  100  syphilitic  families  with  the 
following  results:  In  the  100  families,  331  pregnancies 
occurred  resulting  as  follows:  abortions,  100,  or  30.2  per 
cent;  stillbirths,  31,  or  9.3  per  cent;  living  births,  200,  or 
60.5  per  cent.  Of  the  200  living  births,  39  of  the  children 
had  died  at  the  time  of  the  investigation  and  of  the  161 
living  children,  12  died  during  the  course  of  the  investiga- 
tion. Of  the  161  tested,  107  had  both  clinical  symptoms 
of  syphilis  and  positive  Wassermann  reactions;  5  were 
clinically  positive  but  gave  negative  Wassermann  tests; 
16  were  clinically  negative  but  gave  positive  reactions, 
leaving  33  children  who  were  both  clinically  negative  and 


RESULTS   IN   VARIOUS   STAGES   OF    SYPHILIS  145 

who  gave  a  negative  Wassermann  test.  In  this  series  of 
cases  of  congenital  syphilis  in  young  children  born  of 
syphilitic  parents  Veeder  obtained  96  per  cent  of  positive 
results,  and  his  work  well  illustrates  the  value  of  the  test 
in  the  diagnosis  of  congenital  infection. 

The  Wassermann  test  has  changed  our  conception  of 
Colles'  and  Prof  eta's  laws  and  has  explained  these  appar- 
ent contradictions  in  the  etiology  of  syphilis.  Colles'  law, 
that  an  apparently  healthy  mother  of  a  syphilitic  child 
may  suckle  the  child  with  impunity,  even  though  it  pre- 
sents the  most  infectious  lesions  of  the  disease,  is  ex- 
plained by  the  fact  that  the  mother,  although  showing  none 
of  the  lesions  of  syphilis  is  really  in  the  latent  phase  of 
the  disease,  as  demonstrated  by  the  positive  Wassermann 
reaction  that  is  almost  invariably  obtained  with  the  blood 
of  such  mothers.  Prof  eta's  law,  that  the  child  born  of  a 
syphilitic  mother,  but  presenting  no  evidence  of  the  dis- 
ease, may  suckle  its  mother  with  impunity,  has  also  been 
shown  by  the  Wassermann  test  to  be  due  to  latent  syphilis 
in  the  child,  as  in  these  children  the  test  is  generally  pos- 
itive although  no  symptoms  of  syphilis  are  present  and 
none  may  appear  for  long  periods  of  time.  In  other  words, 
children  born  of  syphilitic  parents  possess  no  immunity 
to  syphilis  as  was  long  believed,  nor  does  a  mother  who 
with  impunity  suckles  her  syphilitic  child,  possess  an  im- 
munity, but  is  herself  syphilitic. 

Just  what  part  late  hereditary  syphilis  plays  in  the  nu- 
merous instances  in  which  a  positive  Wassermann  reaction 
is  found  in  patients  who  deny  any  sj^hilitic  infection  and 
who  may,  or  may  not,  present  evidences  of  the  disease,  is 
still  a  mooted  question.  In  the  writer's  experience  it  has 
not  infrequently  occurred  that  patients  who  are  absolutely 
truthful  and  who  have  given  a  positive  Wassermann  re- 
action, have  denied  even  any  exposure  to  infection  or  any 
lesions  that  could  be  considered  syphilitic,  and  there  has 
been  no  reason  to  doubt  the  truth  of  their  statements.    In 


146  THE   WASSEEMA^Tlsr   TEST 

such  cases,  especially  if  there  are  no  lesions  of  the  dis- 
ease present,  one  should  be  very  careful  to  eliminate  any 
condition  that  has  been  proved  to  give  a  positive  Wasser- 
mann  reaction,  but  after  this  has  been  done  the  explanation 
of  the  reaction  is  often  very  difficult  and  even  impossible. 
That  hereditary  syphilis,  latent  in  character,  is  the  cause 
of  some  of  these  reactions  can  not  be  doubted  but  the 
writer  is  loth  to  believe  that  most  of  them  are  due  to  such 
a  cause.  It  is  more  probable  that  an  accidental  infection 
may  have  occurred  in  infancy  or  childhood,  the  initial  le- 
sion of  which  went  unrecognized,  than  that  the  positive 
test  is  due  to  a  hereditary  infection  that  has  ,never  caused 
symptoms  or  lesions  of  syphilis. 

The  Specificity  of  the  Wassermann  Reaction 

While  it  must  be  admitted  that  the  Wassermann  reac- 
tion is  not  absolutely  specific  for  syphilis,  for  positive 
results  have  sometimes  been  observed  with  other  diseases, 
the  fact  remains  that  a  positive  reaction  with  this  test 
indicates  syphilis  in  so  immense  a  proportion  of  individ- 
uals giving  it  that  the  practical  value  of  the  reaction  in 
the  diagnosis  of  syphilis  is  hardly  at  all  decreased  by  the 
comparatively  few  instances  in  which  such  a  result  is  ob- 
tained in  other  conditions. 

If  one  reads  the  works  of  writers  published  shortly  after 
the  discovery  of  the  Wassermann  test,  the  attention  is  at 
once  called  to  the  large  number  of  conditions  other  than 
syphilis  in  which  positive  reactions  are  reported;  but  if 
these  writings  be  compared  mth  those  published  by  in- 
vestigators during  the  past  five  years,  it  is  evident  that 
most  of  the  so-called  positive  reactions  must  have  been 
due  to  poor  technic,  for  as  our  laiowledge  of  the  technic 
has  increased,  these  nonspecific  reactions  have  decreased 
in  number,  until  today  a  positive  reaction  with  the  test  is 
reported  in  but  few  conditions  that  are  not  due  to  syphilis. 
Any  serologist  who  reports  any  considerable  number  of 


RESULTS   IlSr   VARIOUS   STAGES   OF    SYPHILIS  147 

positive  reactions  with  the  Wassermann  test  in  diseases 
other  than  syphilis  proclaims  that  his  technic  is  poor,  for, 
if  properly  performed,  this  test  will  give  positive  results 
in  very  few  nonsyphilitic  conditions. 

The  earlier  writers  upon  the  subject  reported  positive 
results  in  such  diseases  as  scarlet  fever,  septicemia,  diph- 
theria, carcinoma,  and  most  of  the  acute  infections;  but 
it  is  now  well  knoA^^l  that  these  conditions  do  not  give  a 
positive  reaction  if  the  test  is  properly  performed.  There 
is  still  an  opinion  prevalent  among  the  profession  that 
the  test  is  frequently  positive  in  scarlet  fever,  but  the  work 
of  recent  investigators  proves  conclusively  that  the  test  is 
not  positive  in  this  disease  but  that  when  a  positive  reac- 
tion does  occur  the  child  is  syphilitic  or  syphilis  can  not 
be  excluded.  Hecht,  Lateiner,  and  Wilenko®^  tested  105 
cases  of  scarlet  fever  and  obtained  but  one  positive  result, 
and  Kolmer^^  in  250  cases  which  he  tested  with  both  the 
original  Wassermann  method  and  the  Noguchi  modifica- 
tion obtained  positive  reactions  in  only  5  cases,  or  2  per 
cent,  and  in  these  cases  syphilis  could  not  be  excluded. 
Browning  and  McKenzie^^  examined  37  cases  of  scarlet 
fever  and  did  not  obtain  a  positive  reaction  in  one  of  them. 
These  results  prove  that  the  Wassermann  test  is  not  pos- 
itive in  scarlet  fever  and  that  the  large  proportion  of  pos- 
itive results  reported  in  the  early  literature  of  the  test 
must  have  been  due  to  improper  technic. 

Another  disease  in  which  a  considerable  proportion  of 
positive  reactions  have  been  reported  is  tuberculosis. 
Some  authorities  have  reported  as  high  as  30  to  40  per 
cent  of  positive  results  in  this  disease  but  such  reports  are 
absolutely  unreliable  and  prove  that  the  method  of  per- 
forming the  test  must  have  been  erroneous.  The  writer 
has  tested  hundreds  of  cases  of  tuberculosis  and  while  a 
few  have  shown  a  positive  reaction,  syphilis  could  not  be 
excluded  in  any  case,  and  the  majority  of  the  patients  ad- 
mitted infection.  There  are  many  instances  on  record  of 
syphilis  of  the  lung  existing  along  with  tuberculosis   of 


148  THE   WASSERMANF   TEST 

the  same  organ  and  many  in  which  syphilis  of  the  lung  was 
diagnosed  as  tuberculosis,  and  it  is  these  cases  that  have 
caused  the  impression  that  tuberculosis  frequently  gives 
a  positive  reaction  with  this  test,  together  with  the  large 
number  of  latent  syphilitic  infections  which  are  found  in 
patients  suffering  from  other  diseases.  There  is  also  con- 
siderable evidence  to  prove  that  tuberculosis  renders  an 
individual  much  less  resistant  to  infection  with  Treponema 
pallidum  and  this  probably  has  much  to  do  with  the  prev- 
alence of  the  disease  in  the  tubercular.  However,  that 
tuberculosis  is  a  disease  which  causes  a  positive  Wasser- 
mann  reaction  can  not  be  longer  maintained  in  view  of 
the  negative  results  of  the  best  observers  as  reported  in 
the  most  recent  literature. 

There  are,  however,  a  few  diseases  in  which  a  positive 
Wassermann  reaction  is  sometimes  obtained  and  where 
the  most  careful  examination  fails  to  disclose  any  evi- 
dences of  syphilitic  infection  and  the  most  careful  in- 
quiries any  history  of  the  disease.  These  diseases  are 
yaws  or  frambesia;  the  tubercular  type  of  leprosy;  some 
cases  of  relapsing  fever;  some  malarial  infections,  during 
the  febrile  stage;  and  some  instances  of  experimental  try- 
panosomiasis in  animals.  Of  these,  yaws  is  the  most  im- 
portant because  some  of  the  lesions  of  this  disease  re- 
semble those  of  syphilis  and  in  the  lesions  the  causative 
organism,  Treponema  pertenue,  may  be  found  either  by  a 
dark-field  examination  of  the  secretions  from  the  lesion 
or  by  staining  methods.  As  this  treponema  is  almost 
indistinguishable  morphologically  from  Treponema  pal- 
lidum, and  the  blood  often  gives  a  positive  Wassermann 
reaction,  some  confusion  in  diagnosis  may  result,  but  a 
careful  study  of  the  case  should  enable  the  diagnosis  of 
yaws  to  be  made  from  the  clinical  history  and  symptoms 
alone. 

The  writer  has  observed  positive  reactions  in  five  cases 
of  tertian  malarial  infection,  during  the  febrile  stage  of 
the  paroxysm,   the  blood  becoming  negative   during  the 


RESULTS  IN  VARIOUS  STAGES  OF  SYPHILIS       149 

afebrile  periods.  Thompson^^  states  that  he  has  observed 
positive  results  several  times  in  malarial  disease  during 
the  stage  of  fever  which  became  negative  with  the  disap- 
pearance of  the  fever  and  plasmodia,  and  onr  results  have 
been  confirmed  by  many  observers.  On  the  other  hand, 
the  occurrence  of  a  positive  reaction  in  malaria,  even  dur- 
ing the  paroxysm  of  fever,  is  comparatively  rare,  for  the 
writer  has  examined  many  cases  in  which  the  test  was  neg- 
ative and  other  investigators  report  similar  results.  How- 
ever, the  microscopical  examination  of  the  blood  should 
suffice  to  diagnose  malarial  infections,  so  that  the  occur- 
rence of  a  positive  Wassermann  reaction  should  cause  no 
trouble  in  the  diagnosis  of  the  disease.  If  the  reaction 
persists  after  the  disappearance  of  the  fever  and  Plas- 
modia the  diagnosis  of  syphilis  should  be  made,  for  all 
investigators  report  that  the  positive  reaction  in  malaria 
is  only  temporary  in  character. 

In  the  tubercular  forms  of  leprosy  numerous  positive 
reactions  have  been  reported  but  it  is  impossible  to  say 
just  how  many  of  these  were  true  false  reactions  and  how 
many  were  due  to  a  complicating  syphilitic  infection.  If 
it  be  remembered  that  most  of  the  cases  of  leprosy  that 
have  been  tested  occurred  in  native  races  which  were  thor- 
oughly syphilized  the  difficulty  of  ruling  out  that  infection 
can  be  appreciated.  The  writer  believes  that  a  positive  re- 
action does  occur  in  tubercular  leprosy  in  certain  stages 
of  the  disease  but  he  also  believes  that  the  majority  of 
the  positive  reactions  reported  in  leprosy  have  been  due 
to  a  concurrent  syphilitic  infection.  In  the  anesthetic  form 
of  leprosy  the  Wassermann  test  is  generally  negative,  so 
far  as  reports  indicate.  As  tubercular  leprosy  can  be 
easily  diagnosed  by  the  finding  of  the  bacillus  in  the  le- 
sions a  positive  Wassermann  reaction  should  occasion  no 
confusion  in  the  diagnosis. 

Cases  of  relapsing  fever  have  been  found  to  give  a  posi- 
tive reaction  and  also  some  experimental  infections  with 
trypanosomiasis  but  it  is  yet  unsettled  whether  or  not  the 


150  THE   WASSBRMANN"   TEST 

blood  of  patients  suffering  from  infection  with  Trypano- 
soma gamhiensi  or  other  human  trypanosomes  gives  a  pos- 
itive reaction. 

Positive  reactions  have  been  reported  in  diabetes  mel- 
litus,  in  cases  suffering  from  acidosis,  and  in  pellagra,  but 
so  far  as  the  latter  disease  is  concerned  it  is  now  definitely 
proved  that  the  Wassermann  test  gives  only  negative 
results. 

As  regards  the  number  of  positive  reactions  occurring 
in  diseases  other  than  syphilis  it  may  be  stated  that,  with 
a  properly  performed  and  controlled  method  of  making 
the  test,  it  will  be  so  small  as  to  be  of  practically  no  im- 
portance from  a  diagnostic  standpoint.  The  writer  has  ob- 
tained 12  positive  reactions  among  4,000  individuals  suffer- 
ing from  diseases  other  than  syphilis  or  0.3  of  1  per  cent. 
Of  these  patients  five  were  suffering  from  tertian  malarial 
infection  and  gave  a  positive  result  during  the  febrile 
stage,  which  became  negative  Avhen  the  temperature  re- 
turned to  normal;  three  were  diagnosed  as  tuberculosis; 
three  as  pityriasis  rosea;  and  in  one  the  diagnosis  was 
undetermined.  In  the  pityriasis  rosea  cases  the  reactions 
were  almost  double-plus  (four-plus),  but  would  not  have 
been  reported  as  positive  mthout  a  qualifying  statement, 
while  in  two  of  the  tuberculosis  cases  a  history  of  syphi- 
litic infection  was  afterwards  obtained,  and  the  disease 
could  not  be  excluded  in  the  remaining  case.  But,  even 
admitting  that  all  of  these  cases  gave  a  false  positive  re- 
action, the  real  value  of  the  test  is  but  very  little  affected 
as  a  diagnostic  measure.  Vedder^°  has  obtained  results 
almost  identical  with  those  of  the  writer,  using  the  writ- 
er's modification  of  the  Wassermann  test,  as  he  obtained 
only  four  positive  reactions  among  1,049  individuals  suf- 
fering from  diseases  other  than  syphilis. 

When  it  is  remembered  how  difficult  it  is  to  absolutely 
exclude  a  syphilitic  infection  in  any  individual  and  the 
comparatively  few  instances  in  which  the  Wassermann  test 
gives  a  positive  result  in  diseases  other  than  syphilis,  one 


EBSULTS  IN  VARIOUS  STAGES  OF  SYPHILIS       151 

should  accept  with  great  caution  the  reports  of  positive 
reactions  in  such  diseases,  for  the  experience  of  those  who 
have  worked  with  this  test  for  years  and  in  hundreds  and 
thousands  of  cases  has  been  that  a  positive  reaction,  in  the 
overwhelming  majority  of  instances,  demonstrates  the 
presence  of  syphilis.  There  are  a  very  few  conditions  in 
which  a  positive  reaction  is  obtained  with  this  test  and  in 
which  syphilis  can  be  eliminated,  but  as  most  of  them  can 
be  easily  diagnosed  by  laboratory  and  clinical  methods, 
there  should  be  no  confusion  caused  by  the  result  of  the 
Wassermann  test,  but  even  in  these  cases  a  very  careful 
inquiry  and  examination  should  be  made  before  a  possible 
complication  of  syphilis  is  excluded. 

In  surgical  practice  weak  positive  reactions  are  some- 
times noted  in  blood  sera  collected  shortly  after  ether  and 
chloroform  anesthesia,  so  that  the  test  should  not  be  made 
upon  patients  who  have  been  anesthetized  until  at  least 
two  days  have  elapsed  since  the  anesthetic  was  admin- 
istered. 

In  conclusion,  it  may  be  stated  that  the  substance  or 
substances  that  cause  a  positive  Wassermann  reaction  ap- 
pear to  be  practically  peculiar  to  the  blood  serum  of  pa- 
tients suffering  from  syphilitic  infection,  and  while  it  can 
not  be  claimed  that  the  test,  when  positive,  is  absolutely 
specific  of  syphilis,  from  a  practical  standpoint  it  is  doubt- 
ful if  a  more  specific  test  is  employed  in  medicine,  the 
margin  of  error  appearing  to  be  less  than  five-tenths  of 
one  per  cent. 


CHAPTER  VIII 

THE  INTERPRETATION  OF  THE  RESULTS  OF  THE 
WASSERMANN     TEST.      THE    WASSERMANN 
TEST    AS    AN    INDEX    OF    THE    PREVA- 
LENCE OF  SYPHILIS  IN  COMMUNITIES 

The  writer  has  often  been  impressed  with  the  evident 
misconceptions  of  many  practitioners  of  medicine  regard- 
ing the  nature  of  the  AVassermann  reaction,  the  percentage 
of  positive  results  that  may  be  expected  in  the  various 
stages  of  the  disease,  but,  especially  the  exact  significance 
that  may  be  attached  to  a  positive  or  negative  result.  In 
his  experience,  many  patients  have  been  told  by  their  med- 
ical attendant  that  they  were  free  from  syphilis,  the  state- 
ment being  based  upon  a  single  negative  result  with  this 
test,  while  others  have  been  told  that  they  were  suffering 
from  the  disease  upon  the  strength  of  a  plus  or  plus-minus 
reaction,  in  the  absence  of  either  a  history  of  infection  or 
of  any  symptoms  of  the  disease. 

Such  interpretations  of  the  results  of  the  Wassermann 
test  are  unwarranted  and  have  brought  the  test  into  dis- 
repute in  certain  localities.  In  addition,  innocent  individ- 
uals have  suffered  great  mental  anguish  from  being  un- 
justly stigmatized  as  afflicted  with  syphilis,  and  others 
have  been  infected  by  those  who  have  been  told  that  they 
were  free  from  the  disease  by  some  careless  or  ignorant 
practitioner.  Even  today,  almost  ten  years  since  the  Was- 
sermann test  has  been  in  general  use  by  the  profession, 
there  is  a  surprising  amount  of  ignorance  regarding  the 
interpretation  of  the  various  grades  of  the  reaction,  as  re- 
ported by  different  laboratories,  and  much  of  the  fault 
lies  with  the  laboratory  reports,  for  the  terms  strongly 
positive,  positive,  and  weakly  positive,  so  commonly  used, 

152 


INTERPRETATIOE"    OF   RESULTS  153 

are  misleading,  unless  they  are  carefully  explained  in  ref- 
erence to  each  case.  In  fact,  the  writer  has  always  believed 
that  only  three  terms  should  be  used  in  reporting  the  re- 
sults of  the  Wassermann  test ;  i.  e.,  positive,  doubtful,  and 
negative,  leaving  it  to  the  clinician  to  decide  how  much 
weight  should  be  attached  to  any  grade  of  the  reaction 
which  is  reported  as  doubtful,  and  which,  in  the  army, 
would  mean  any  degree  of  the  reaction  less  than  complete 
inhibition  of  hemolysis.  While  there  is  no  doubt  that  many 
cases  of  syphilis  react  weakly  consistently,  especially  in  the 
early  and  late  latent  stages,  never  presenting  complete  in- 
hibition of  hemolysis,  the  writer  believes  that  all  cases  show- 
ing less  than  complete  inhibition  should  be  reported  as 
doubtful,  and  the  interpretation  of  the  reaction  left  with  the 
clinician.  Of  course,  if  there  is  a  clear  history  of  infection 
and  typical  clinical  signs  are  present,  the  doubtful  reaction 
would  at  once,  in  the  opinion  of  the  clinician,  be  considered 
as  positive  and  as  supporting  the  diagnosis  of  syphilis,  but 
if,  on  the  other  hand,  there  was  no  history  of  infection  and 
no  S3niiptoms  are  present,  a  doubtful  Wassermann  reac- 
tion is  not  sufficient  proof  upon  which  to  base  a  diagnosis 
of  syphilitic  disease. 

The  statements  here  m^ade  regarding  the  interpretation 
of  the  results  of  the  Wassermann  test  are  based  upon  over 
50,000  tests  personally  performed  by  the  writer,  the  major- 
ity of  them  being  reexaminations  made  as  a  control  of  the 
treatment  of  the  disease,  the  remainder  being  upon  patients 
presenting  the  symptoms  common  to  the  various  stages  of 
syphilis.  The  percentage  of  positive  and  negative  results 
obtained  in  these  cases  has  already  been  given  (Chapter 
VII),  as  well  as  the  terminology  used  in  reporting  the  re- 
sults, but  it  is  well  here  to  repeat  the  terminology  used 
in  the  army  laboratories. 

Four  degrees  of  the  reaction  are  noted  in  reports  from 
the  army  laboratories.  A  positive  reaction  is  reported  as 
double-plus  (+  +)  and  means  that  there  was  absolute  inhibi- 
tion of  hemolysis.     A  doubtful  reaction    is    reported    as 


154  THE   WASSEEMAXX   TEST 

plus  (-)  or  iDlus-ininus  (--).  the  former  term  indicating 
that  there  was  over  50  per  cent  inhibition  of  hemolysis,  the 
latter  that  there  was  less  than  50  per  cent  inliibition  of  hem- 
olysis. A  negative  reaction  is  reported  as  minus  (-).  In 
most  ci^-ilian  laboratories  the  results  of  the  TTassermann 

test  are  reported  as  four-plus   ( ),  three-plus  (+  +  ^), 

two-plus  (--),plus  (-^), plus-minus  (--),  and  negative  (-). 
The  four-plus  reaction  corresponds  to  the  army  double- 
plus;  the  tliree-plus  and  hvo-plus  to  the  army  plus;  and  the 
Ijlus  and  plus-minus  to  the  army  plus-minus. 

These  distinctions  between  the  terminology  used  should 
be  borne  in  mind  in  the  following  discussion  of  the  inter- 
pretation of  the  results  of  the  test. 

The  v,Titer  has  already  discussed  the  specificity  of  the 
"Wassermann  reaction  (Chapter  ^T!I)  and  there  remains  the 
question  as  to  the  significance  of  the  positive  reaction. 
Some  authors  have  denied  that  a  positive  reaction  with  this 
test  indicates  the  presence  of  syphilis,  claiming  that  after 
the  disease  disappears  the  reaction  still  persists,  and  that 
the  positive  result  is  simply  an  indication  that  the  patient 
has  had  syphilis  sometime  in  the  past.  In  the  light  of  mod- 
ern research  upon  the  disease  tliis  theory  can  no  longer  be 
considered  as  worthy  of  credence,  for  it  is  the  unanimous 
experience  of  those  who  have  had  the  most  to  do  -^vith  the 
treatment  of  syphilis,  as  controlled  by  the  Wassermann 
test,  that  T\-ith  the  disappearance  of  the  treponema  and  of 
sA^nptoms  the  reaction  also  disappears  and  that,  in  cases 
that  are  really  cured,  the  reaction  remains  permanently 
absent.  That  this  is  true  has  been  proved  experimentally 
upon  infected  animals,  and  the  disappearance  of  the  reac- 
tion in  these  animals  after  the  administration  of  salvarsan 
which  has  caused  the  disappearance  of  the  treponemas,  is 
conclusive  evidence  that  the  positive  reaction  does  not  per- 
sist after  the  cure  of  the  sypliilitic  infection. 

There  are  still  others  who  maintain  that,  in  the  absence 
of  symptoms,  a  positive  "Wassermann  reaction  does  not  in- 
dicate SA-philis.    To  those  who  still  hold  to  this  opinion,  the 


IFTERPKETATION"    OF   EESULTS  155 

writer  lias  only  to  say  that  all  experience  lias  sliOA\-n  that, 
if  the  few  conditions  in  which  a  positive  result  sometimes 
occurs  can  be  eliminated,  a  positive  reaction  means  the 
presence  sometvliere  in  the  tody  of  living  treponemas  and 
that  the  question  of  history  or  symptoms  should  have  no 
influence  in  deciding  the  advisability  of  treatment.  It  is 
just  this  class  of  cases  that  later  furnish  the  great  bulk  of 
our  patients  suffering  from  paresis,  tabes,  aortitis,  aneu- 
rism, and  syphilitic  disease  of  the  viscera,  the  latter  often 
only  discovered  at  autopsy.  In  cases  in  which  there  is 
good  reason  to  question  tlie  possibility  of  syphilis  the  pa- 
tient's blood  serum  should  be  titrated,  for,  as  Vedder^^ 
well  says: 

''One-tenth  of  a  cubic  centimeter  of  some  sera  is  re- 
quired to  give  a  double-plus  reaction,  while  one-thousandth 
of  a  cubic  centimeter  of  another  serum  may  be  sufficient 
to  give  a  double-plus  reaction.  There  would  be  little  hes- 
itation in  making  a  diagnosis  of  syphilis  in  the  latter  case 
whether  clinical  SATuptoms  were  present  or  not.  There 
might  be  doubt  in  the  former  case." 

As  the  interpretation  of  the  results  of  the  test  varies  in 
the  different  stages  of  the  disease,  each  stage  lAdll  be  con- 
sidered separately. 

The  Interpretation  of  the  Results  of  the  Wassermann 
Test  in  the  Primary  Stage  of  Syphilis. — It  is  in  this  stage 
of  the  disease  that  the  weaker  grades  of  reaction  are  of 
most  value  from  a  diagnostic  standpoint.  As  would  be  ex- 
pected, the  earlier  in  the  primary  stage  the  blood  is  tested, 
the  larger  will  be  the  percentage  of  negative  results  and 
also  the  larger  the  percentage  of  doubtful  reactions.  As 
a  certain  amount  of  complement-binding  substance  must 
be  present  to  give  a  positive  result,  the.  longer  the  infection 
has  lasted  the  greater  the  chance  that  this  amount  is  pres- 
ent and  that  the  test  wiU  be  positive.  This  fact  has  al- 
ready been  showm  in  the  table  giving  the  date  of  the  ap- 
pearance of  the  positive  Wassermann  reaction  in  600  cases 
of  syphilis,  but,  with  a  clear  iiistory  of  infection  and  a 


156  THE   WASSERMANN   TEST 

suspicious  lesion,  it  is  proper  to  consider  as  positive,  in 
this  stage  of  the  disease,  reactions  that  would  be  always 
doubtful  at  other  stages. 

A  plus-minus  reaction  (plus  of  most  laboratories) 
should  be  considered  as  a  negative  reaction,  in  most  in- 
stances, but  if  such  a  reaction  is  obtained  within  a  week 
or  two  after  the  appearance  of  the  initial  lesion  it  may  be 
considered  as  of  some  confirmatory  value,  but  never  as 
diagnostic  of  syphilis. 

A  plus  reaction  (+),  the  double-plus  (++),  and  three-plus 
(+  +  +)  reaction  of  other  laboratories,  when  obtained  in  the 
primary  stage  of  syphilis,  may  be  interpreted  in  practice 
as  a  positive  reaction,  provided  there  is  a  history  of  ex- 
posure and  a  suspicious  lesion  is  present.  The  writer 
often  obtains  this  type  of  reaction  in  cases  in  which  the 
initial  lesion  has  lasted  for  from  one  to  three  or  four  weeks, 
and  he  has  never  seen  a  plus  reaction  in  such  cases  become 
negative,  except  as  the  result  of  treatment,  and  if  un- 
treated, the  patients  invariably  developed  secondary  symp- 
toms. In  the  absence  of  a  history  of  infection  or  the  initial 
lesion  a  plus  reaction  should  not  be  considered  as  diag- 
nostic of  syphilis. 

A  double-plus  (++)  reaction,  the  four-plus  (+  +  +  +)  re- 
action of  some  laboratories,  is  always  diagnostic  of  syph- 
ilis in  this,  as  in  every  other  stage  of  the  disease,  provided 
the  other  conditions  that  sometimes  give  such  a  reaction, 
can  be  eliminated.  This  type  of  reaction,  which  means  ab- 
solute inhibition  of  hemolysis,  in  the  writer's  experience 
has  occurred  in  14  per  cent  of  the  cases  of  primary  syph- 
ilis tested  during  the  first  week  after  the  appearance  of 
the  initial  lesion;  in  22  per  cent  tested  during  the  second 
week;  in  41  per  cent  tested  during  the  third  week;  in  53 
per  cent  tested  during  the  fourth  week ;  and  in  61  per  cent 
of  cases  tested  during  the  fifth  week  after  the  appearance 
of  the  initial  lesion.  Eeactions  so  strong  as  to  almost  equal 
complete  inhibition  of  hemolysis  occur  in  a  large  numbef  of 


INTERPRETATTOIsr    OF   RESULTS  157 

cases  of  primary  syphilis  and  these  should  be  reported  as 
double-plus  or  four-plus  reactions.  In  these,  the  super- 
natant salt  solution,  after  the  undissolved  corpuscles  have 
settled  to  the  bottom  of  the  test  tube,  shows  a  slight  red- 
dish yellow  tinge,  demonstrating  that  a  small  amount  of 
hemoglobin  has  been  dissolved  from  the  erythrocytes,  but 
practically  such  reactions  are  positive  and  should  be  re- 
ported as  such. 

A  negative  reaction,  in  the  primary  stage  of  syphilis,  is 
of  no  value  whatever  in  eliminating  syphilis.  Even  though 
it  persists  until  the  initial  lesion  disappears  and  during 
the  latent  stage  before  the  occurrence  of  secondary  symp- 
toms, it  can  not  be  relied  upon  as  evidence  that  syphilis 
does  not  exist.  As  the  writer  has  already  shown,  at  least 
10  per  cent  of  patients  suffering  from  primary  syphilis 
will  give  a  negative  A¥assermann  reaction,  so  that,  in  the 
presence  of  a  suspicious  lesion,  the  negative  reaction  is 
of  no  value,  and  the  patient  should  never  be  assured,  upon 
the  strength  of  such  a  reaction,  that  his  symptoms  are  not 
due  to  syphilis. 

As  already  emphasized,  every  suspicious  lesion  follow- 
ing exposure,  whether  situated  upon  the  penis  or  else- 
where, should,  if  possible,  be  examined  with  the  dark-field 
apparatus  for  Treponema  pallidum.  This  should  never  be 
neglected  and  a  Wassermann  test  made  in  preference,  for 
the  dark-field  examination  is  the  diagnostic  method  of 
choice  during  this  stage  of  the  disease,  but  Avhere  such  an 
examination  can  not  be  made,  or  where  it  gives  negative 
results,  a  Wassermann  test  should  always  be  performed. 
The  writer  has  observed  many  instances  in  which  it  was 
impossible  to  demonstrate  Treponema  pallidum,  either  by 
the  dark-field  apparatus  or  in  stained  preparations  made 
from  the  suspicious  lesion,  but  in  which  the  Wassermann 
test  was  positive  and  thus  the  diagnosis  was  established. 
In  extraurethral  chancres  the  test  has  been  of  the  greatest 
value  in  enabling  us  to  make  an  early  diagnosis  and  in  the 
latent  period,  after  the  healing  of  the  initial  lesion  and  be- 


158  THE   WASSERMANN   TEST 

fore  the  appearance  of  secondary  symptoms,  the  Wasser- 
mann  test  is  the  only  method  available  for  making  a  diag- 
nosis. It  is  in  this  latter  class  of  cases,  where  there  is 
only  a  history  of  a  healed  lesion  to  assist  one,  that  the  test 
is  of  the  very  greatest  clinical  value  in  diagnosis  and  every 
such  case  should  be  tested  repeatedly,  if  possible,  as  treat- 
ment applied  before  the  appearance  of  secondary  s>mip- 
toms  is  much  more  efficient  than  when  it  is  administered 
afterward. 

The  Interpretation  of  the  Results  of  the  Wassermann 
Test  in  the  Secondary  Stage  of  Syphilis. — In  the  secondary 
stage  of  syphilis  a  positive  result  is  obtained  in  from  95 
to  96  per  cent  of  all  cases,  so  that  in  this  stage  of  syphilis 
the  Wassermann  test  may  be  stated  to  be  almost  always 
confirmatory  of  the  clinical  findings.  HoAvever,  from  4  to 
5  per  cent  of  secondary  cases,  in  the  writer's  experience, 
will  give  a  negative  reaction,  even  when  repeatedly  tested, 
and  when  marked  secondary  lesions  are  present,  so  that 
the  mere  absence  of  a  positive  reaction  is  no  proof  that 
the  patient  is  not  suffering  from  the  disease. 

A  douhle-plus  (four-phis)  reaction  in  patients  present- 
ing the  lesions  of  secondary  syphilis  or  lesions  that  are 
suspicious  of  this  stage  of  the  disease,  is  conclusive  evi- 
dence of  the  presence  of  s^^philis  under  the  limitations  al- 
ready mentioned  in  discussing  this  same  type  of  reaction 
in  the  primary  stage ;  i.  e.,  if  the  conditions  which  some- 
times give  a  positive  reaction  can  be  eliminated  in  the 
particular  ease  examined. 

A  plus  {tivo-  or  three-plus)  reaction  is  not  of  as  much 
value  in  the  secondary  stage  of  syphilis  as  in  the  primary, 
but  even  in  this  stage  this  type  of  reaction  occurs  in  about 
10  per  cent  of  cases,  in  the  writer's  experience,  and  when 
accompanied  by  suspicious  s^nnptoms,  should  be  inter- 
preted as  a  positive  reaction.  In  the  majority  of  such  in- 
stances the  reaction  is  equal  to  the  three-plus  reaction 
reported  by  most  laboratories,  inhibition  of  hemolysis  be- 
ing almost  complete,  but  even  if  the  reaction  is  less  than 


INTEKPRETATION   OF   RESULTS  159 

this  it  should  be  regarded  as  confirmatory  of  the  presence 
of  the  disease  in  patients  presenting  clinical  symptoms  or 
a  clear  history  of  a  primary  lesion. 

A  plus-minus  {plus  or  plus-minus)  reaction  in  the  sec- 
ondary stage  of  syphilis  is  valueless  from  a  diagnostic 
standpoint.  In  these  cases  the  diagnosis  Avill  have  to  rest 
upon  the  clinical  symptoms  present  or  upon  the  history  of 
the  case.  On  the  other  hand,  the  presence  of  this  type  of 
reaction  should  not  lead  to  any  hope  that  the  disease  is 
not  present,  as  some  secondary  infections  never  give  more 
than  a  plus-minus  reaction. 

A  negative  reaction  when  suspicious  s^Tnptoms  of  sec- 
ondary syphilis  are  present  is  of  greater  value  than  at  any 
other  stage  of  the  disease,  but  it  should  be  remembered 
that  practical^  five  per  cent  of  cases  of  secondary  syph- 
ilis give  a  negative  reaction  and  that  many  of  these  cases 
remain  negative  for  weeks  and  months.  It  is  only  when 
the  negative  reaction  persists  for  several  months  and  no 
symptoms  of  syphilis  are  present  during  this  time  that  it 
may,  with  justice,  be  regarded  as  conclusive  evidence  of 
the  absence  of  syphilis.  On  the  other  hand,  if  the  case 
examined  gives  no  history  of  a  primary  lesion  and  the 
symptoms  present  are  only  slightly  suspicious,  a  negative 
reaction  which  persists  for  two  or  three  months  may  be 
regarded  as  proving  the  absence  of  syphilis,  provided 
treatment  has  not  been  administered  in  the  meanwhile. 

The  Interpretation  of  the  Results  of  the  Wassermann 
Test  in  the  Tertiary  Stage  of  Syphilis. — In  the  tertiary 
stage  of  syphilis,  when  definite  tertiary  lesions  are  present, 
the  Wassermann  test  gives  as  high  a  percentage  of  posi- 
tive results  as  in  the  secondary  stage,  but  there  are  many 
cases  tested  in  this  stage  in  which  the  symptoms  are  atyp- 
ical or  only  slightly  suspicious,  or  in  which  the  nervous 
system  alone  is  involved,  which  give  weak  or  negative  re- 
actions, so  that  the  total  percentage  of  positive  results  in 
this  stage  of  the  disease  is  slightly  below  that  encountered 
in  the  secondary  stage.    In  many  of  these  cases,  also,  spe- 


160  THE   WASSERMANN   TEST 

cific  treatment  has  been  administered  and  although  not 
successful  in  preventing  the  development  of  lesions,  it  may 
have  reduced  markedly  the  strength  of  the  Wassermann 
reaction. 

A  douhle-plus  (four-plus)  reaction  with  the  test  in  the 
tertiary  stage  of  syphilis  is  diagnostic  of  the  disease  under 
the  limitations  already  noted  in  discussing  the  interpreta- 
tion of  this  type  of  reaction  in  the  primary  and  secondary 
stages. 

A  plus  [tivo-plus  or  three-plus)  occurs  more  frequently 
in  this  stage  of  syphilis  than  in  the  secondary  stage  and  is, 
therefore,  of  greater  diagnostic  value.  In  patients  pre- 
senting suspicious  lesions  of  this  stage,  especially  in  those 
in  which  there  is  apparent  involvement  of  the  central  nerv- 
ous system,  the  writer  believes  that  this  type  of  reaction 
should  be  given  the  same  diagnostic  value  as  the  double- 
plus  (four-plus)  reaction,  with  the  same  limitations. 

A  plus-minus  {tivo-plus  and  plus-minus)  reaction,  unless 
permanent,  or  treatment  has  been  administered,  is  of  no 
value  in  the  diagnosis  of  syphilis  during  this  stage  of  the 
disease.  In  those  instances  in  which  there  is  no  history  of 
infection  and  the  symptoms  present  are  merely  slightly 
suspicious,  this  type  of  reaction  should  be  considered  as 
negative,  provided  it  persists  over  two  or  three  months,  but 
in  the  presence  of  a  clear  history  of  infection,  especially  if 
treatment  has  been  administered,  a  plus-minus  reaction 
should  not  lead  one  to  consider  the  case  as  nonsyphilitic. 

A  negative  reaction  in  patients  suspected  of  tertiary 
syphilis  is  of  no  value  in  excluding  the  disease  unless  it 
persists  for  several  months  and  S3rmptoms  have  not  de- 
veloped meanwhile.  In  the  absence  of  a  history  of  infec- 
tion and  of  symptoms  which  are  typical,  a  negative  re- 
action may  be  interpreted  as  excluding  syphilis,  provided 
no  specific  treatment  has  been  administered  and  it  remains 
negative  for  a  period  of  three  months.  In  all  suspected 
tertiary  cases,  when  the  Wassermann  test  is  negative,  a 
test  should  be  made  upon  the  cerebrospinal  fluid. 


INTEEPRETATION    OF    RESULTS  161 

The  Interpretation  of  the  Results  of  the  Wassermann 
Test  in  the  Latent  Stages  of  Syphilis. — The  results  of  the 
Wassermann  test  differ  in  the  early  and  late  latent  cases 
but  the  difference  is  not  sufficient  to  justify  our  classify- 
ing the  cases  into  early  and  late  cases,  so  far  as  the  inter- 
pretation of  the  results  is  concerned.    In  the  early  latent 
cases,  that  is,  the  cases  tested  during  the  latent  period  be- 
tween the  healing  of  the  initial  lesion  and  the  occurrence 
of  secondary  symptoms,  a  higher  percentage  of  positive 
results  is  obtained  than  in  latent  periods  occurring  later 
in  the  disease,  and  the  lowest  percentage  of  positive  re- 
sults is  obtained  in  latent  cases  in  which  the  period  of 
latency  occurs  after  the  development  of  secondary  or  ter- 
tiary lesions.    The  percentage  of  positive  results  obtained 
by  the  writer  in  latent  infection,  including  both  early  and 
late  cases,  has  been  68  per  cent,  so  that  in  this  class  of 
infections  one  must  expect  about  30  per  cent  of  negative 
results.    One  of  the  most  important  reasons  for  the  lower 
percentage  of  positive  results  in  latent  cases  is  that  the 
vast  majority  of  these  patients  have  received  more  or  less 
specific  treatm^ent  and  in  most  of  the  cases  the  test  was 
made  in  order  to  determine  the  result  of  this  treatment. 
Therefore,  it  follows  that  the  plus  reaction  will  have  more 
diagnostic  value  in  these  treated  cases  than  in  other  stages 
of  syphilis  when  treatment  has  not  been  given,  and  that 
a  negative  reaction  will  have  less  value  in  excluding  the 
disease. 

A  double-plus  (four-plus)  reaction  occurs  less  frequently 
in  latent  cases  than  in  other  stages  of  syphilis,  but  when  it 
is  obtained  it  is  absolutely  diagnostic  of  the  disease  under 
the  limitations  already  mentioned. 

A  plus  (Uvo-plus  or  three-plus)  reaction,  in  latent  cases, 
where  there  is  a  clear  history  of  infection,  should  be  given 
the  same  interpretation  as  the  double-plus  (four-plus)  re- 
action, especially  if  specific  treatment  has  been  admin- 
istered.   It  is  the  writer's  belief  that  this  type  of  reaction 


162  THE   WASSERMANN"   TEST 

in  latent  cases,  under  the  conditions  named,  is  diagnostic 
of  syphilitic  infection. 

A  plus-minus  (plus  or  plus-minus)  reaction  in  latent  in- 
fections, when  there  is  a  clear  history  of  infection,  symp- 
toms have  been  present  in  the  past,  or  specific  treatment 
has  been  given,  should  aronse  the  suspicion  that  the  disease 
is  still  present,  but  the  test  should  be  repeated  two  or 
three  times,  with  a  similar  result,  before  a  diagnosis  of 
syphilis  should  be  entertained.  In  such  cases  a  plus-minus 
reaction  usually  means  insufficient  treatment,  and  indicates 
that  further  treatment  is  required.  In  the  absence  of  a  his- 
tory of  infection,  or  of  previous  symptoms,  a  diagnosis  of 
syphilis  should  never  be  made  upon  a  plus-minus  reaction. 

A  negative  reaction  in  cases  suspected  of  being  in  the 
latent  stage  of  syphilis  has  no  value  as  excluding  the  dis- 
ease unless  it  is  consistently  negative  over  several  months, 
in  the  absence  of  specific  treatment  and  of  symptoms  of 
the  disease.  A  negative  result  is  obtained  in  at  least  30 
per  cent  of  latent  syphilitics  so  that  it  has  little  value  in 
excluding  the  disease. 

Interpretation  of  the  Results  of  the  Wassermann  Test  in 
Syphilitic  Disease  of  the  Central  Nervous  System. — The  in- 
terpretation of  the  results  of  the  Wassermann  test  in  syph- 
ilitic disease  of  the  central  nervous  system  is  considered 
in  Chapter  X  of  this  work. 

Interpretation  of  the  Results  of  the  Wassermann  Test 
after  Specific  Treatment. — The  interpretation  of  the  results 
obtained  in  patients  who  have  received  specific  treatment  is 
considered  in  Chapter  IX  of  this  work. 

General  Summary. — From  the  writer's  experience  he  be- 
lieves that  the  following  general  conclusions  regarding  the 
interpretation  of  the  results  of  the  Wassermann  test  are 
justified : 

1.  If  the  diseases,  other  than  syphilis,  that  sometimes 
give  a  positive  result  with  the  Wassermann  test,  can  be 
excluded,  a  double-plus  (four-plus  of  many  writers)  re- 
action is  absolutely  diagnostic  of  syphilis.    It  is  believed 


INTERPRETATION    OF   RESULTS  163 

that,  under  such  conditions,  this  type  of  reaction  is  specific 
of  the  disease,  whether  symptoms  are  present  or  not,  or 
whether  there  is  or  is  not  a  history  of  infection. 

2.  Under  the  same  conditions,  a  plus  reaction  (three- 
plus  or  two-plus  of  many  writers)  may,  in  primary,  ter- 
tiary, and  latent  infections  be  regarded  as  diagnostic,  pro- 
vided there  is  a  clear  history  of  infection,  or  suspicious 
clinical  symptoms  are  present.  In  secondary  cases  this 
type  of  reaction  is  not  diagnostic  unless  typical  secondary 
symptoms  are  present.  In  the  absence  of  either  history 
or  symptoms  this  type  of  reaction  should  not  be  regarded 
as  diagnostic  of  syphilis. 

3.  A  diagnosis  of  syphilis  should  never  be  made  upon  a 
plus-minus  reaction  alone.  Many  perfectly  normal  individ- 
uals give  this  type  of  reaction  at  times  in  their  blood  serum. 
In  latent  infections  a  plus-minus  reaction,  if  persistent, 
should  be  followed  by  further  specific  treatment. 

4.  A  single  negative  reaction,  where  there  is  no  history 
of  infection  and  where  symptoms  are  not  present,  is  of  con- 
siderable value  as  a  corroborative  sign  that  syphilis  is  not 
present,  but  where  there  is  any  suspicion  that  the  disease 
may  be  present  it  has  very  little  value  in  excluding  syphilis. 
The  experiments  already  mentioned  upon  the  variation  in 
the  complement-binding  power  of  the  blood  serum  of  known 
syphilitics  illustrate  the  truth  of  this  statement,  and  it  is 
only  when  a  negative  reaction  is  repeatedly  obtained  over 
a  long  period  of  time  that  it  can  be  considered  as  good 
evidence  of  the  absence  of  the  disease,  and  even  then  the 
spinal  fluid  should  be  tested  and,  if  possible,  a  provocative 
Wassermann  test  should  be  made  upon  the  blood.  The 
same  remarks  apply  to  cases  that  have  been  given  specific 
treatment,  a  negative  reaction  being  of  no  value  as  indi- 
cating a  cure  of  the  infection,  unless  the  reaction  remains 
persistently  negative  over  a  period  of  at  least  one  year. 
In  such  cases  a  provocative  Wassermann  test  should  be 
made,  and  the  spinal  fluid  should  be  tested.    A  luetin  test 


164  THE   WASSERMAlSriSr   TEST 

should  also  be  made  in  certain  cases  before  the  patient  is 
discharged  as  cured. 

The  interpretation  of  the  results  of  the  Wassermann 
test  must  rest  very  largely  with  the  clinician,  for  the  clin- 
ical picture  present  is  often  more  decisive  than  is  the  re- 
sult of  the  test,  and  it  is  the  clinician's  place  to  reconcile 
the  result  of  the  test  with  the  clinical  picture,  rather  than 
the  serologist's.  The  latter  should  simply  report  what 
actually  occurs  when  the  patient's  blood  serum  is  tested, 
without  any  reference  whatever  to  the  clinical  history  or 
the  symptoms  >vhich  may  be  present,  and  it  remains  with 
the  clinician  to  interpret  the  report  in  the  light  of  his 
patient's  condition.  The  writer  believes  that  much  harm 
has  been  done  by  the  serologist  allowing  the  history  of  a 
case,  or  the  symptoms  present,  to  influence  his  reading  of 
the  Wassermann  test,  and  it  is  alw^ays  better  to  have  the 
test  read  without  the  serologist  knowing  anything  what- 
ever about  the  history  or  symptoms  of  the  patients  from 
whom  the  specimens  were  collected. 

The  Wassermann  Test  as  an  Index  of  the  Prevalence 

of  Syphilis 

Since  the  discovery  of  the  Wassermann  test  it  has  been 
largely  used  in  determining  the  prevalence  of  syphilis  in 
communities,  hospitals,  and  among  various  classes  of  peo- 
ple, and  has  been  of  the  greatest  service  in  demonstrating 
how  widespread  this  disease  really  is,  and  the  compara- 
tively large  number  of  people  who  suffer  from  it  in  nearly 
all  countries  and  among  nearly  all  classes  of  society.  In 
fact,  it  may  be  stated,  that  before  the  days  of  the  Wasser- 
mann reaction,  although  it  was  generally  recognized  that 
syphilis  was  a  very  prevalent  disease  in  many  countries, 
we  had  a  very  poor  conception  of  its  real  prevalence,  espe- 
cially in  the  United  States,  so  that  from  a  sociological 
standpoint,  the  test  has  been  of  such  value  as  to  almost 
revolutionize  our  ideas  regarding  the  subject. 


INDEX  OF  THE  PREVALENCE  OF  SYPHILIS        165 

It  is  now  well  known,  and  has  been  demonstrated  time 
and  again  by  Wassermann  surveys  of  the  class  mentioned, 
that  between  80  and  90  per  cent  of  prostitutes  who  have 
pursued  their  calling  for  as  long  as  five  years  or  more, 
are  syphilitic,  although  it  should  be  remembered,  in  con- 
sidering this  high  percentage,  that  only  a  comparatively 
small  percentage  of  the  women  were  actually  capable  of 
transmitting  the  infection  at  the  time  the  tests  were  made, 
so  far  as  clinical  evidence  was  obtainable.  However,  in  a 
disease  like  syphilis  that  has  its  periods  of  infectivity  and 
quiescence,  this  high  percentage  of  syphilitics  among  pros- 
titutes furnishes  evidence  enough  of  the  danger  of  illicit 
sexual  intercourse,  and  one  often  wonders  why  the  disease 
is  not  much  m.ore  common  than  it  appears  to  be  in  the 
usual  professional  experience.  As  a  matter  of  fact,  syph- 
ilis is  much  more  common,  as  shown  by  the  results  of  the 
Wassermann  test,  than  usually  supposed  and,  in  this  coun- 
try as  well  as  in  others,  is  pretty  well  distributed  through 
all  classes. 

As  a  result  of  the  application  of  the  Wassermann  test 
in  Wassermann  surveys,  as  they  are  called,  a  great  mass 
of  evidence  has  been  accumulated  regarding  the  preva- 
lence of  syphilis  in  different  communities  and  among  dif- 
ferent classes  of  the  population.  Thus,  Southard^^  per- 
formed 6,000  Wassermann  tests  in  the  Harvard  Neuro- 
pathologic  Laboratory  and  found  23  per  cent  positive; 
Hammond,®^  who  applied  the  test  to  both  patients  and 
others  at  the  New  Jersey  State  Hospital,  found  6.3  per 
cent  positive ;  Thompson''*  made  1,000  tests  at  the  Arkansas 
State  Hospital  for  Nervous  Diseases  and  found  33  per 
cent  positive ;  Eosenberger^^  made  5,106  Wassermann  tests 
upon  the  patients  at  the  Philadelphia  General  Hospital, 
during  1916,  and  found  27.4  per  cent  positive;  and  John- 
son,^*' who  examined  by  means  of  the  Wassermann  test 
224  children  shomng  anemia  and  malnutrition,  found  that 
over  33  per  cent  gave  a  positive  Wassermann  reaction. 

There  is  no  better  place  to  study  the  prevalence  of  syph- 


166 


THE   WASSERMANN   TEST' 


ills  than  among  the  personnel  of  the  army,  for  recruits 
are  draAvn  from  all  classes  of  society  for  the  service  and 
from  all  parts  of  the  United  States.  The  most  important 
and  conclusive  contribution  to  our  knowledge  of  the  preva- 
lence of  syphilis  in  the  army,  and,  consequently,  in  civil 
life,  has  been  made  by  Lieut.  Colonel  Vedder,  who  pub- 
lished his  results  in  1915,^^  and  from  whose  work  I  shall 
quote  largely  in  considering  this  subject. 

In  his  work,  Vedder  considered  that  all  cases  giving  a 
double-plus  (four-plus)  reaction  were  without  doubt  syph- 
ilitic, while  those  giving  a  plus  (three-plus  reaction)  were 
probably  syphilitic.  Summarized,  his  results  are  presented 
in  Table  XXXIV: 

Table  XXXIV 
Occurrence  of  Syphilis  in  Army,  from  Vedder 


ESTIMATED 

CLASSES 

TOTAL 

tested 

KNOWN 

CASES 

% 

WAS- 

SER- 

MANN 

% 

undoubt- 
ed SYPH- 

ilitics 

% 

WAS- 
SER- 

MANN 
% 

TOTAL 

NUMBER  OF 

PROBABLE 

SYPH- 

ILITICS 

% 

Recruits 

1,019 

0 

1.1^ 

7.75 

9.02 

16.77 

Cadets 

621 

0 

'i.fil 

2.57 

2.89 

5.46 

White  enlisted 

1,577 

3.44 

4:.n 

8.21 

7.87 

16.08 

Colored  enlisted 

1,472 

1.08 

21.80 

22.21 

13.11 

36.00 

Porto  Rico  Regt. 

531 

13.55 

28.58 

42.37 

13.55 

55.93 

Military  convicts 

1,145 

6.48 

9.50 

15.98 

5.67 

21.65 

Insane  soldiers 

567 

3.51 

8.29 

11.80 

7.41 

19.21 

Tuberculous  soldiers 

229 

7.56 

15.72 

23.28 

15.72 

39.00 

Soldiers  home 

1,171 

11.62 

13.40 

25.02 

9.73 

34.75 

There  may  be  some  question  as  to  whether  the  cases 
giving  a  plus  reaction  examined  by  Vedder  should  be 
called  probably  positive,  but,  even  if  these  cases  are  ex- 
cluded, the  figures  given  in  the  table  are  very  striking 
and  show  that  syphilis  is  a  far  more  prevalent  disease  than 
is  generally  supposed  by  the  average  practitioner. 

Among  the  recruits  examined  by  Vedder,  numbering 
1,019,  a  double-plus  (four-plus)  reaction  was  obtained  in 
7.75  per  cent,  or  practically  8  per  cent.     These  men  had 


INDEX  OF  THE  PREVALENCE  OF  SYPHILIS        167 

passed  a  most  thorough  physical  examination  for  admis- 
sion to  the  army  and  presumably  presented  no  evidences 
of  syphilis  when  accepted.  If  to  this  number  be  added 
the  9.02  per  cent  of  cases  giving  a  plus  (three-plus)  re- 
action, the  total  percentage  of  syphilitics  would  amount 
to  16.77  per  cent.  These  recruits  were  distributed  through 
the  areas  of  the  United  States  covered  by  the  recruiting 
stations  at  Fort  Slocum,  New  York,  and  Columbus  Bar- 
racks, Ohio,  and  represented  numerous  occupations  before 
enlistment.  Vedder  concludes  that  his  observations,  to- 
gether with  the  known  figures  for  rejection  for  syphilis  of 
applicants  for  admission,  show  that  practically  "among 
young  men  in  civil  life  between  the  ages  of  20  and  30,  and 
of  the  general  class  belonging  to  the  occupations  mentioned, 
the  percentage  of  syphilis  may  be  estimated  at  at  least 
16.77  per  cent,  and  there  is  good  reason  for  believing  that 
it  is  fully  20  per  cent. ' ' 

The  writer's  own  observations  upon  the  percentage  of 
positive  Wassermann  reactions  obtained  in  recruits  for 
the  army,  numbering  several  thousands  of  cases,  is  prac- 
tically the  same  as  Vedder 's,  as  regards  the  occurrence  of 
double-plus  reactions,  the  percentage  obtained  by  the 
writer  being  8.64  per  cent  instead  of  7.75.  It  is  not  be- 
lieved that  the  cases  giving  a  plus  reaction  should  be  con- 
sidered as  positive  unless  there  is  a  history  of  infection 
or  of  suspicious  symptoms  in  the  past;  but  in  the  writer's 
experience  such  a  history  may  be  obtained  upon  careful 
questioning  in  enough  of  the  cases  to  raise  the  percentage 
of  s^^hilis  among  recruits  of  the  army  to  10  per  cent  when 
they  are  admitted  to  the  service.  If  to  these  are  added 
the  percentage  of  rejections  for  syphilis,  based  upon  actual 
symptoms  of  the  disease,  the  writer  believes  that  at  least 
15  per  cent  of  all  applicants  for  enlistment  for  the  army, 
prior  to  the  present  war,  were  infected  vdth  syphilis,  and 
this  means  that  practically  15  per  cent  of  the  average  male 
civilian  population  of  this  country  between  the  ages  of 
20  and  30  are  infected.    This  appears  to  be  an  enormous 


168  THE  wasserma:n"n  test 

percentage  but  the  figures  are  borne  out  in  Wassermann 
surveys  upon  a  similar  class  of  men,  as  regards  age,  in 
other  countries,  and  in  surveys  made  in  our  large  hospitals 
upon  all  classes  of  cases  admitted  to  them. 

Vedder's  results  upon  the  class  of  young  men  who  have 
entered  West  Point  are  important,  for  these  young  men 
come  from  the  average  middle  class  family  of  this  coun- 
try, have  been  well  educated,  and  must  be  above  the  aver- 
age in  intelligence  in  order  to  pass  the  rigid  examinations 
for  entrance  to  the  militars^  academ}'.  In  this  class  of 
young  men  Vedder  found  2.57  per  cent  of  double-plus 
(four-plus)  reactions  among  the  621  cadets  tested,  and 
2.89  per  cent  of  plus  reactions.  If  these  be  added  together, 
there  is  a  total  percentage  of  over  5  per  cent  which  he  con- 
siders as  probably  s^^Dhilitic.  The  writer  believes  that 
probably  three  per  cent  would  be  about  the  amount  of 
syphilis  in  this  class  of  cases,  and,  applied  to  a  similar 
class  of  3^oung  men  in  civilian  life,  (the  class  attending  our 
colleges)  it  would  mean  that  about  this  percentage  of  col- 
lege men  are  syphilitic  before  entering  college. 

As  regards  the  amount  of  syphilis  in  white  and  colored 
enlisted  men  in  the  army,  Vedder  found  that  among  the 
white  enlisted  men  8.21  per  cent  gave  a  double-plus  (four- 
plue)  reaction  while  among  the  colored  enlisted  men  no 
less  than  22  per  cent  gave  this  type  of  reaction.  His  fig- 
ures as  regards  the  greater  percentage  of  syphilitic  infec- 
tion among  the  colored  men  of  the  army  agree  with  those  of 
many  investigators  who  have  made  Wassermami  surveys 
upon  the  colored  race  in  this  country. 

Vedder's  results  as  to  the  prevalence  of  syphilis  among 
the  patients  at  the  army  hospital  for  tuberculosis,  at  Fort 
Bayard,  N.  M.,  are  of  interest,  as  they  dem.onstrate  that 
a  much  larger  proportion  of  tuberculous  patients  give  a 
positive  reaction  than  patients  suffering  from  other  dis- 
eases. He  found  that  no  less  than  23.28  per  cent  of  the 
229  patients  examined  gave  a  positive  reaction  and  he 
states  that  his  results  are  almost  identical  with  those  of 


INDEX   OF   THE   PREVALENCE    OF   SYPHILIS  169 

Letulle,  Bergeron,  and  Lepine  in  Paris,  who  found  that 
one  out  of  every  five  tuberculous  patients  was  also  syph- 
ilitic. These  results  explain  fully  the  large  number  of 
positive  reactions  which  some  writers  have  claimed  are  due 
to  tuberculosis,  but  the  most  careful  investigations  have 
shown  that  tuberculosis  per  se  does  not  give  a  positive 
reaction  with  the  Wassermann  test,  so  that  those  patients 
who  do  react  positively  are  undoubtedly  suffering  from  a 
coincident  syphilitic  infection. 

The  writer,  while  working  upon  his  method  of  comple- 
ment fixation  in  the  diagnosis  of  tuberculosis,  had  the 
opportunity  of  testing  166  patients  in  the  General  Hospital 
at  Fort  Bayard  and  found  that  nearly  10  per  cent  of  them 
gave  a  positive  reaction  with  the  Wassermann  test,  while 
about  6  per  cent  were  known  to  be  syphilitic,  giving  a  total 
percentage  of  16  per  cent  of  syphilitics  in  the  number  ex- 
amined. While  these  figures  are  not  as  large  as  those 
recorded  by  Vedder  they  amply  demonstrate  the  fact  that 
syphilis  and  tuberculosis  are  more  often  associated  than 
are  other  diseases,  and  that  one  may  expect  a  large  per- 
centage of  syphilitic  infection  among  the  tuberculous. 

Irvine,^^  as  the  result  of  various  Wassermann  surveys, 
concludes  that  from  10  to  15  per  cent  of  the  population  of 
the  State  of  Minnesota  are  syphilitic,  or  from  200,000  to 
300,000  people.  He  states  that  Wassermann  surveys 
made  in  the  asylum  at  Fergus  Falls  showed  that  30  per 
cent  of  the  inmates  were  syphilitic  and  that  the  average 
of  many  similar  surveys  is  20  per  cent.  Peterson^"  found 
that  among  477  obstetric  patients,  no  less  than  5.8  per 
cent  were  syphilitic,  and  of  606  gynecologic  patients  the 
test  was  positive  in  8.2  per  cent.  Regarding  the  value  of 
the  Wassermann  test  in  obstetric  and  gynecologic  prac- 
tice he  says:  ''In  many  of  the  cases,  both  where  there  was 
a  history  of  infection  and  when  this  was  lacking,  physical 
examination  in  the  gynecologic  clinic  failed  to  arose  sus- 
picion of  the  presence  of  syphilis.  The  point  to  be  empha- 
sized is  that  the  same  careful  physical  examinations  have 


170  THE   WASSERMANN   TEST 

been  made  in  this  particular  clinic  for  years  with  only 
an  occasional  patient  suspected  of  being  luetic.  When, 
however,  such  examinations  are  reinforced  by  routine  Was- 
sermann  examinations,  nearly  10  per  cent  of  the  patients 
are  found  syphilitic."  Ladd"°  found  that  25.5  per  cent  of 
patients  applying  for  treatment  at  the  dispensaries  of  the 
Washington,  D.  C,  Casualty  and  Asylum  Hospitals  gave 
a  double-plus  (four-plus)  Wassermann  reaction,  and  that 
of  these  14.6  per  cent  were  colored,  and  10.9  per  cent  white 
patients. 

An  entire  volume  could  be  written  covering  the  statis- 
tics of  the  almost  innumerable  Wassermann  surveys  that 
have  been  made  in  different  localities  but  the  illustrations 
here  given  demonstrate  the  value  of  the  test  in  the  study  of 
the  prevalence  of  syphilis.  In  fact,  no  longer  is  any  data 
of  scientific  value  regarding  this  subject  unless  it  is  based 
upon  the  results  of  carefullj^  performed  Wassermann  re- 
actions, for  the  day  has  passed  when  clinical  e\adence  alone 
is  to  be  considered  as  sufficient  upon  which  to  base  figures 
regarding  the  prevalence  of  syphilis  in  a  community  or 
country.  The  cases  of  syphilitic  infection  that  are  appar- 
ently without  clinical  sj^uptoms  far  outnumber  those  that 
present  typical  symptoms  and  the  only  way  that  we  at  pres- 
ent possess  of  making  a  diagnosis  in  these  latent  infections 
is  by  means  of  the  Wassermann  test.  Aside  from  the  value 
of  this  test  in  the  diagnosis  of  the  disease  and  in  the  scien- 
tific control  of  its  treatment,  perhaps  the  greatest  value 
of  the  test  is  in  the  demonstration  of  the  extent  to  which 
this  insidious  disease  has  permeated  society,  an  extent  al- 
together unsuspected  before  the  use  of  the  reaction  in  Was- 
sermann surveys. 


CHAPTER  IX 

THE  EFFECT  OF  TREATMENT  UPON  THE  WAS- 
SERMANN  REACTION.    THE  WASSERMANN 
TEST  AS  A  CONTROL  OF  THE  TREAT- 
MENT OF  SYPHILIS.     THE  PRO- 
VOCATIVE WASSERMANN 
REACTION 

The  treatment  of  sypliilis,  either  with  mercury  or  with 
salvarsan  or  neosalvarsan  often  has  a  marked  effect  npon 
the  results  of  the  Wassermann  test,  rendering  strongly 
positive  reactions  negative  or  greatly  lessening  the  strength 
of  the  reaction  in  those  cases  in  which  a  positive  result  is 
not  rendered  negative.  This  fact  has  rendered  the  use  of 
the  test  invaluable  as  a  guide  to  the  efficiency  of  the  treat- 
ment of  the  disease  mth  various  drugs  and  has  enabled 
the  profession  to  discard  many  drugs  vaunted  as  specifics 
for  the  disease  but  which  have  been  proved  by  the  Was- 
sermann test  to  be  absolutely  worthless.  One  of  the  most 
striking  examples  of  this  fact  is  the  instance  of  sodium 
cacodylate,  which  at  one  time  was  announced  as  almost  the 
equal  of  salvarsan  in  the  treatment  of  syphilis,  but  which 
was  soon  proved  by  the  Wassermann  test  to  be  practically 
worthless,  so  far  as  curing  the  infection,  and  which  is  not 
used  at  the  present  time  in  the  treatment  of  the  disease 
except  by  those  ignorant  of  the  work  which  has  been  done 
proving  its  worthlessness  or  those  willfully  blind  to  the  re- 
sults of  that  work. 

The  Wassermann  reaction  is  generally  conceded  to  be 
the  most  reliable  and  delicate  of  all  of  the  symptoms  of 
syphilis  and  for  this  reason  the  effect  of  treatment  upon 
the  reaction  is  of  the  utmost  practical  importance.    A  posi- 

171 


172  THE   WASSERMAFN   TEST 

tive  reaction,  as  has  been  noted,  means  the  presence  in  the 
body  of  living  treponemas,  and  as  long  as  the  reaction 
remains  positive,  just  so  long  is  the  patient  a  victim  of 
syphilitic  infection,  regardless  of  the  occurrence  of  other 
s^Tuptoms  of  the  disease.  The  effect  of  treatment  with 
various  drugs  can  be  easily  followed  in  the  case  of  syph- 
ilis by  frequently  repeated  Wassermann  tests,  and  in  this 
way  one  can  secure  a  reliable  picture  of  the  results  of  any 
particular  form  of  treatment.  In  view  of  the  importance 
of  this  subject  the  follomng  chapter  contains  the  personal 
observations  of  the  writer  upon  the  effect  upon  the  Was- 
sermann reaction  of  treatment  ^^dth  salvarsan  and  mer- 
cury, which  have  been  previously  published,^"  together 
with  remarks  upon  the  test  as  a  control  of  the  treatment 
of  syphilis,  and  upon  the  provocative  Wassermann  reaction. 

The  Effect  of  Treatment  upon  the 
Wassermann  Reaction 

Prior  to  1911  there  was  little  in  the  literature  upon  the 
effect  of  treatment  on  the  Wassermann  reaction  but  since 
that  time  a  great  deal  of  data  has  been  published  show- 
ing that  the  reaction  is  markedly  influenced  by  treatment 
A\ith  certain  drugs,  and  that  positive  reactions  often  be- 
come negative  and  remain  so  for  considerable  periods  of 
time  after  the  cessation  of  treatment.  Unfortunately,  in- 
vestigation has  also  shoA\Ti  that  the  early  treatment  with 
mercury  and  salvarsan  was  insufficient  and  that  most  of 
the  cases  so  treated  relapsed  within  a  year  after  treat- 
ment was  stopped,  so  that  at  the  present  time  no  intelligent 
practitioner  believes  that  a  few  injections  of  mercury  or  of 
salvarsan  or  neosalvarsan  \\t.11  cure  syphilis. 

The  Effect  upon  the  Reaction  of  Treatment  with  Salvar- 
san.— The  data  given  in  regard  to  the  effect  upon  the  Was- 
sermami  test  of  treatment  mth  salvarsan  was  collected  by 
the  writer  between  1910  and  1913  and  is  based  upon  a  com- 
paratively few  injections  of  the  drug,  as  Ehrlich's  idea 


THE    EFPECT    OF    TREATMENT  1(6 

that  one  or  two  injections  of  this  drug  would  sterilize  the 
body,  so  far  as  Treponema  pallidum  was  concerned,  was 
still  the  belief  and  hope  of  the  profession.  However,  the 
facts  deduced  from  these  experiments  and  observations 
have  since  been  fully  confirmed  and  the  data  demonstrate 
the  effect  upon  a  positive  Wassermann  reaction  of  treat- 
ment with  this  drug. 

Among  those  who  had  contributed  valuable  data  regard- 
ing the  effect  of  salvarsan  upon  the  Wassermann  reaction 
prior  to  the  writer's  publications  may  be  mentioned  the 
following : 

Nichols  and  Fordyce"-  treated  11  patients  with  salvarsan 
with  the  following  results  upon  the  Wassermann  reaction: 
In  8  the  reaction  became  negative,  1  in  19  days;  2  in  21 
days ;  1  in  23  days ;  1  in  25  days ;  1  in  39  days ;  1  in  3  months ; 
and  1  in  3  months  and  11  days.  Michaelis^"^  treated  110 
patients  and  states  that  the  reaction  may  become  negative 
within  a  period  from  2  to  10  weeks  after  the  injection. 
Fordyce"*  states  that  the  results  of  the  Wassermann  test 
after  salvarsan  have  varied  within  wide  limits,  in  his  expe- 
rience, but  in  general  a  change  occurred  in  from  4  to  5 
weeks.  The  earliest  change  from  a  positive  to  a  negative 
reaction  observed  by  him  Avas  in  6  days  after  the  injec- 
tion. He  states  that  ''several  cases  showed  alternating 
negative  and  positive  phases."  Noguchi"^  analyzed  very 
carefully  102  cases  treated  with  salvarsan,  more  than  half 
of  them  being  under  observation  for  over  three  months 
while  the  remainder  had  been  injected  four  weeks  pre- 
viously. He  made  quantitative  examinations  of  their  blood 
serum  for  complement-binding  power,  the  tests  being  made 
before  the  injection  of  salvarsan,  and  at  intervals  of  one 
day,  three  days,  one  week,  two  weeks,  three  weeks,  four 
weeks,  six  weeks,  eight  weeks,  etc.,  after  the  injection.  He 
found  that  30  cases  became  negative,  24  reduced  to  less 
than  1  antibody  unit,  while  the  remaining  48  cases  still 
contain  more  than  1  antibody  unit,  giving  strong  positive 
reactions.    In  40  per  cent  of  the  primary  cases,  in  37  per 


174 


THE   WASSEKMANN   TEST 


cent  of  the  secondary  cases,  in  35  per  cent  of  tertiary,  in 
33  per  cent  of  latent,  in  14  per  cent  of  hereditary,  and  in 
50  per  cent  of  incipient  tabes  the  reaction  became  nega- 
tive. The  average  of  the  negative  reactions  equalled  33.7 
per  cent  of  the  total  102  cases.  Of  the  negative  cases  re- 
ported by  Noguchi,  34  in  number,  3  became  negative  in 
two  weeks,  10  in  three  weeks,  11  in  four  weeks,  5  in  five 
weeks,  4  in  six  weeks,  and  1  in  seven  weeks. 

Animal  Experiments. — The  effect  upon  the  Wassermann 
reaction  of  treatment  with  salvarsan  can  be  shown  in  rab- 
bits experimentally  infected  with  syphilis.  Through  the 
kindness  of  Major  H.  J.  Nichols,  the  writer  had  the  oppor- 
tunity of  repeatedly  testing  the  blood  serum  of  rabbits  ex- 
perimentally infected  with  syphilis  and  yaws,  and  Tables 
XXXV  and  XXXVI  illustrate  the  gradual  increase  in  the 
strength  of  the  Wassermann  test  in  these  animals  after  in- 
fection, and  the  disappearance  of  the  positive  reaction  after 


Table  XXXV 

Eesults  of  Wassermann  Test  before  and  after  Treatment  with  Salvarsan 

IN  Rabbit  Infected  with  Syphilis* 


AMOUNT  OF 
BLOOD 

DATE  OP  TESTS 

SERUM,  CO. 

Jan.  23 

Jan. 27 

Jan.  30 

Feb.  6 

Feb.  8 

Feb.  10 

Feb.  13 

Feb. 18 

0.05 

_ 

+ 

+ 

+  + 

_ 

+ 

0 

_ 

0.10 

_ 

+  + 

+  + 

+  + 

+  - 

+  + 

_ 

_ 

0.15 

0 

+  + 

+  + 

+  + 

+  - 

+  + 

0 

- 

0.20 

+  + 

0 

+  + 

+  + 

+  - 

+  + 

- 

- 

*Treponema  pallidum  found  in  testicular  lesion  Jan.    19. 
administered  intravenously  Feb.   6. 


0.02  gram  salvarsan  per  kilo 


Table  XXXVI 

Results  of  Wassermann  Test  before  and  after  Treatment  with  Salvarsan 

IN  Rabbit  Infected  with  Yaws* 


amount  of 
blood 

DATE  OF  TESTS 

serum,  C.C, 

Jan.  23 

Jan.  30 

Feb.  2 

Feb.  6 

Feb.  8 

Feb.  10 

Feb.  13 

Feb. 17 

0.05 
0.10 

0.15 
0.20 

- 

+  - 

+ 

+  + 

+  + 
+  + 

+  + 
+  + 
+  + 

+  + 

+  - 

+  - 

4-- 
+ 

- 

- 

^Treponema,  pertenue  found  in  lesion  Jan.   21.      0.02   gram  salvarsan  per  kilo  adminis- 
tered intravenously   Feb.   6. 


THE   EFFECT   OF   TREATMENT  175 

treatment  with  salvarsan.  The  sign  (++)  indicates  com- 
plete inhibition  of  hemolysis. 

In  the  rabbit  infected  with  syphilis  the  treponema  was 
found  in  the  lesion  on  January  19,  but  the  Wassermann 
test  did  not  become  positive,  even  mth  0.2  c.c.  of  the  serum, 
until  February  6.  One  week  after  the  injection  of  salvar- 
san the  reaction  had  become  negative  and  the  table  shows 
that  it  had  begun  to  diminish  in  strength  two  days  after 
the  injection. 

The  results  of  treatment  with  salvarsan  upon  the  rab- 
bit infected  with  yaws  v/ere  quite  similar,  the  reaction  be- 
coming negative  one  week  after  the  intravenous  injection 
of  the  drug.    ' 

The  following  summary  of  the  writer's  observation  upon 
the  effect  of  treatment  with  salvarsan  on  the  Wassermann 
reaction  embraces  500  cases  which  were  observed  and 
tested  for  at  least  four  months  after  treatment.  Of  these 
500  cases  only  288  became  negative,  or  57.6  per  cent,  but 
these  results  were  due  to  the  prevalence,  at  that  time,  of  giv- 
ing but  one  or  two  intravenous  injections  of  salvarsan  in- 
stead of  several,  the  intramuscular  method  of  using  the  drug 
giving  altogether  the  best  results  as  regards  the  disappear- 
ance of  the  positive  reaction. 

The  real  effect  of  treatment  with  salvarsan  upon  the 
"Wassermann  reaction  can  only  be  accurately  understood 
if  several  factors  are  taken  into  consideration,  the  most 
important  being  the  stage  of  syphilis  during  which  the 
drug  is  administered;  the  intensity  of  the  positive  Was- 
sermann reaction  before  treatment;  the  method  of  admin- 
istration of  the  drug  and  the  dose  administered;  as  well 
as  the  amount  and  kind  of  previous  mercurial  treatment. 

The  Stage  of  Syphilis  in  Relation  to  the  Disappearance 
of  the  Reaction. — The  results  of  the  writer  have  shown 
that  regardless  of  the  method  of  administration  or  the 
dose,  the  stage  of  the  disease  has  much  to  do  with  the 
effect  of  treatment  with  salvarsan  upon  the  AVassermann 
reaction.    This  is  shown  in  Table  XXXVII. 


176 


THE   WASSERMANN"   TEST 


Table  XXXVII 

The  Eesults  of  Treatment  with  Salvarsan  upon  the  Wassermann  Test  in 
THE  Various  Stages  of  Syphilis 


stage  of  disease 

total  cases 

became  negative 

remained  positive 

number 

% 

number 

% 

Primary 
Secondary 
Tertiary 
Latent 

95 

285 
52 
68 

63 

169 

17 

39 

66.3 
59.3 
32.6 
57.3 

32 

116 

35 

29 

33.6 
40.7 
67.3 
42.6 

totals 

500 

288 

57.6 

212 

42.4 

An  analysis  of  this  table  is  interesting.  In  95  primary- 
cases  63  became  negative,  or  66.3  per  cent;  in  285  second- 
ary cases,  169,  or  59.3  per  cent  became  negative;  in  52 
tertiary  cases  17  or  32.6  per  cent  became  negative ;  and  in 
68  latent  cases,  including  both  early  and  late  latent  infec- 
tions, 35,  or  57.3  per  cent  became  negative. 

From  the  above  it  is  evident  that  the  effect  upon  the 
Wassermann  reaction  of  treatment  with  salvarsan  is  most 
marked  during  the  primary  stage  of  the  disease,  as  would 
be  expected  and  least  marked  during  the  tertiary  stage, 
and  that  the  longer  the  infection  has  lasted  the  less  is  the 
effect  upon  the  reaction  of  the  treatment. 

Time  of  Disappearance  of  the  Reaction  in  Relation  to  the 
Stage  of  Syphilis 

The  rapidity  with  which  the  positive  Wassermann  re- 
action disappears  after  treatment  with  salvarsan  depends 
largely  upon  the  stage  of  the  disease  as  is  shown  in  Table 
XXXVIII. 

T.\BLE  XXXVIII 

The  Relation  of  the  Stage  of  Syphilis  to  the  Time  of  Disappearance  of 
the  Wassermann  Reaction,  after  Treatment  with  Salvarsan 


stage  of 
disease 

total 

CASES 

1 

WEEK 

2 
WEEKS 

3 

WEEKS 

4 
WEEKS 

5 

WEEKS 

6 

WEEKS 

7 

WEEKS 

8 
WEEKS 

Primary 
Secondary 
Tertiary 
Latent 

63 

169 

17 

39 

6 

13 

3 

2 

25 

44 

6 

9 

20 

39 
2 

7 

8 
33 

5 
10 

2 

17 

0 

6 

2 

13 

1 

5 

0 

5 
0 
0 

0 

5 

0 
0 

TOTALS 

288 

24 

84 

68 

56 

25 

21 

5 

5 

THE   EFFECT   OF   TREATMENT  177 

Regarding  the  time  of  disappearance  of  the  reaction 
after  treatment  with  salvarsan  it  will  be  noted  that  the 
best  results  were  obtained  in  the  primary  stage  of  the  dis- 
ease, no  less  than  51  of  the  63  primary  cases  that  became 
negative  becoming  so  mthin  three  weeks  and  the  longest 
time  after  treatment  in  which  the  reaction  became  negative 
in  this  stage  was  six  weeks.  In  the  secondary  stage  the 
reaction  disappeared  generally  dnring  the  second,  third, 
or  fourth  weeks,  the  longest  period  being  eight  weeks.  In 
the  tertiary  stage  the  results  appear  more  favorable  than 
in  the  primary,  1  of  the  17  cases  becoming  negative  within 
three  weeks  but  the  number  of  cases  is  too  small  to  base 
such  an  assertion  upon,  and  the  writer  believes  that  the 
evidence  is  sufficient  to  prove  that  the  best  results  are 
obtained  in  the  primary  stage  of  syphilis. 

As  will  be  noted,  none  of  the  reactions,  in  any  stage  of 
the  disease,  became  negative  after  eight  weeks,  so  that  it 
would  appear  that  a  positive  reaction  which  remains  so 
after  a  period  of  eight  weeks  has  elapsed  since  the  last 
salvarsan  treatment,  should  always  be  an  indication  for 
further  treatment.  If  the  drug  is  given  intravenously,  as 
it  generally  is  today,  the  period  may  be  shortened  to  less 
than  this,  but  it  is  believed  that  eight  weeks  is  a  practical 
and  safe  period  to  wait  in  order  to  ascertain  if  the  Was- 
sermann  reaction  will  become  negative. 

The  Relation  of  the  Intensity  of  the  Reaction  to  Its  Dis- 
appearance after  Salvarsan.^ — The  stronger  the  Wasser- 
mann  reaction  the  less  is  it  affected  by  treatment  with 
salvarsan,  as  is  shown  in  Table  XXXIX.  In  this  table 
and  those  that  follow,  it  should  be  remembered  that  a 
double-plus  (++)  reaction  means  absolute  inhibition  of 
hemolysis,  or  the  four-plus  reaction  of  most  writers ;  a  plus 
(+)  reaction  at  least  50  per  cent  of  inhibition,  or  the  three- 
and  double-plus  reaction  of  some  laboratories ;  and  a  plus- 
minus,  less  than  50  per  cent  of  inhibition. 

An  anal^^sis  of  this  table  shows  that  of  295  cases  in  which 
the    Wassermann    reaction    was    double-plus    (four-plus). 


178 


THE   WASSERMAIsriT   TEST 


Table  XXXIX 

The  Relation  op  the  Intensity  of  the  Wassermann  Eeaction  to  its 
Disappearance  after  Treatment  with  Salvarsan 


character 

OF 

TOTAL 
CASES 

BECAME  NEGATIVE 

REMAINED  POSITIVE 

reaction 

NUMBER 

% 

NUMBER 

% 

+  + 
+ 

+  - 

295 

169 

36 

145 
110 

33 

41.9 
65.0 
91.6 

150 
59 

3 

50.8 

34.9 

8.3 

500 

288 

57.6 

212 

42.4 

41.9  per  cent  became  negative;  of  169  giving  a  plus  (three 
or  two  plus)  reaction,  65  per  cent  became  negative,  and  of 
36  cases  showing  a  plus-minus  reaction,  91  per  cent  be- 
came negative.  As  regards  the  latter  cases  it  may  be  stated 
that  they  were  either  very  early  primary  cases  or  cases  that 
had  previously  received  specific  treatment  to  a  greater  or 
lesser  extent.  It  will  thus  be  seen  that  the  poorest  results 
as  regards  the  effect  of  treatment  upon  the  Wassermann 
test  were  obtained  in  cases  shomng  complete  inhibition  of 
hemolysis  and  the  results  gradually  grew  better  with  the 
loss  in  the  strength  of  the  reaction. 

The  Relation  of  the  Stage  of  Syphilis  and  the  Intensity 
of  the  Wassermann  Reaction  to  its  Disappearance  after 
Treatment  with  Salvarsan. — The  percentage  of  negative 
results  obtained  by  treatment  with  salvarsan  in  each  stage 
of  syphilis  is  considerably  influenced  by  the  intensity  of 
the  reaction.  Table  XL  illustrates  the  effect  of  the  inten- 
sity of  the  reaction  upon  its  disappearance  in  each  stage 
of  the  disease. 

From  this  table  it  is  evident  that  the  effect  of  treatment 
with  salvarsan  upon  the  Wassermann  test,  in  every  stage 
of  syphilis,  varies  with  the  intensity  of  the  reaction,  being 
most  pronounced  in  the  cases  showing  the  weaker  reactions 
and  least  in  those  giving  complete  inhibition  of  hemolysis. 
From  these  data  it  is  evident  that  the  earlier  treatment  is 
begun  in  syphilis  the  better  will  be  the  results,  for  the 
cases  of  tertiary  syphilis,  for  instance,  which  gave  a  double- 
plus  reaction,  gave  a  very  low  percentage  of  negative  re- 


THE   EFFECT   OF   TREATMENT 


179 


Table  XL 

The  Relation  of  the  Stage  of  Syphilis  and  the  Intensity  of  the  Wasser- 

MANN  Reaction  to  Its  Disappearance  after  Treatment  with 

Salvarsan 


STAGE  OF 

CHARACTER 
■ 
OF    REACTION 

TOTAL 

CASES 

BECAME  NEGATIVE 

REMAINED 

POSITIVE 

SYPHILIS 

NUMBER 

% 

NUMBER 

% 

Primary 
(95  cases) 

+  + 
+ 

+  - 

44 
41 
10 

24 
31 

8 

54.5 
75.6 
80.0 

20 

10 

2 

45.4 
24.3 
20.0 

Secondary 
(285  cases) 

+  - 

185 
86 
14 

98 
58 
13 

52.9 
67.4 

92.8 

87 

28 

1 

47.0 

32.5 

7.1 

Tertiary 
(52  eases) 

+  + 
+ 

+  - 

33 

IG 

3 

5 

9 
3 

15.1 

56.2 
100.0 

28 
7 
0 

84.8 

43.7 

0.0 

Latent 

(68  cases) 

+  + 

+  - 

33 

26 

9 

18 

12 

9 

54.5 

46.1 

100.0 

15 

14 
0 

45.4 

53.8 

0.0 

suits  after  treatment;  i.  e.,  only  15  per  cent  and  even  in 
the  cases  giving  a  plus  reaction  the  percentage  of  negative 
reactions  was  only  a  little  over  50  per  cent.  It  will  be  ob- 
served that  in  the  latent  stage  a  higher  percentage  of  cases 
showing  a  donble-plus  reaction  became  negative  than  of 
those  showing  a  pins  reaction,  but  this  was  due  to  previous 
mercurial  treatment  in  a  larger  proportion  of  the  cases 
giving  a  double-plus  reaction. 

From  these  data  it  is  also  evident  that  the  intensity  of 
the  Wassermann  reaction  in  the  various  stages  of  syphilis 
is  of  considerable  prognostic  importance.  ^Vhile  in  all  of 
the  stages  of  syphilis,  with  the  exception  of  the  latent  stage, 
the  results  were  better  in  cases  giving  a  plus  reaction  than 
in  those  giving  a  double-plus,  the  difference  is  most  marked 
in  the  tertiary  stage  of  the  disease  and  least  in  the  pri- 
mary. As  regards  the  double-plus  cases,  the  best  results 
were  obtained  in  the  primary  and  latent  stages  of  syphilis 
and  the  poorest  results  in  the  tertiary  stage.  The  good  re- 
sults in  the  latent  stage  may  be  explained  by  the  fact  that 
many  of  these  cases  were  early  latent  cases,  for  the  late  la- 
tent cases  are  much  more  resistant  to  treatment.  Of  the 
cases  giving  a  plus  reaction  the  best  results  were  obtained  in 


180 


THE   WASSERMANN   TEST 


the  primary  stage,  the  poorest  in  the  latent  stage,  while 
the  results  in  the  secondary  stage  were  better  than  those 
in  the  tertiary.  The  prognosis,  as  regards  the  disappear- 
ance of  the  reaction,  is  most  unfavorable  in  the  tertiary 
cases  giving  a  double-plus  (four-plus)  reaction. 

The  Relation  of  the  Method  of  Administration  of  Salvar- 
san  to  the  Disappearance  of  the  Wassermann  Reaction  ta 
Treated  Cases. — The  method  of  administration  of  salvar- 
san  has  a  very  marked  influence  upon  the  effect  of  the  drug 
on  the  Wassermann  reaction.  Of  the  500  cases  studied, 
209  were  treated  by  the  intramuscular  injection  of  the  alka- 
line solution,  249  by  the  intravenous  injection  of  the  drug, 
and  42  by  combined  intramuscular  and  intravenous  injec- 
tions. Table  XLI  illustrates  the  effect  of  the  method  of 
administration  upon  the  AA'^assermann  reaction. 


Table  XLI 

The  Methods  op  Administration  of  Salvarsan  in  Relation  to  the 
Disappearance  of  the  Wassermann  Reaction  in  Treated  Cases 


method  of 

TOTAL 
CASES     . 

BECAME 

NEGATIVE 

REMAINED   POSITIVE 

administration 

NUMBER 

% 

NUMBER 

% 

Intramuscular 

Intravenous 

Combined 

209 

249 

42 

159 
101 

28 

76.0 
44.5 
66.6 

50 
148 

14 

24 

55.5 

33.3 

totals 

500 

288 

57.6 

212 

42.4 

It  will  be  observed,  from  a  study  of  the  table,  that  the 
effect  of  intramuscular  injections  of  salvarsan  upon  the 
reaction  is  much  more  pronounced  than  other  methods  of 
administration.  Unfortunately,  this  method  of  adminis- 
tration has  been  almost  abandoned  by  the  profession  and  in 
1he  army  it  has  been  entirely  replaced  by  the  intravenous 
method,  owing  to  the  pain  and  complications  that  followed 
the  injections  and  the  time  lost  in  hospital.  However, 
there  is  no  question  that  the  intramuscular  method  is  in- 
finitely more  efficient  in  treatment  than  the  intravenous,  as 
shown  by  the  results  upon  the  Wassermann  test,  for  of  the 
209  cases  so  treated,  most  of  them  receiving  but  one  injec- 
tion of  0.6  gram  of  salvarsan,  159,  or  76  per  cent  became 


THE   EFFECT   OF   TREATMENT  181 

negative,  and,  as  will  be  noted  later,  in  discussing  the  test 
as  a  control  of  treatment,  fewer  cases  treated  by  the  intra- 
muscular method  relapsed  than  by  the  intravenous. 

The  figures  given  for  the  intravenous  method  are  below 
what  they  should  be  for  the  reason  that  the  majority  of 
the  cases  studied  only  received  one  intravenous  injection. 
Further  observations  upon  this  method  of  administration 
and  its  effect  upon  the  Wassermann  reaction  have  shown 
that  with  from  three  to  five  intravenous  injections  the  re- 
sults are  as  good  as  those  obtained  with  the  intramuscular 
injections,  but  the  data  here  given  are  sufficient  to  show 
that  the  Wassermann  reaction  can  be  rendered  negative  by 
only  one  intravenous  injection  in  at  least  40  per  cent  of  the 
cases. 

The  effect  upon  the  reaction  of  the  combined  intra- 
muscular and  intravenous  method  of  using  salvarsan  was 
determined  in  42  cases,  of  which  66  per  cent  became  nega- 
tive, thus  indicating  that  the  results  were  not  as  good  with 
the  combined  method  as  with  the  intramuscular  method 
alone.  However,  this  result  was  due  to  the  fact  that  a  num- 
ber of  the  intramuscular  cases  had  two  injections  of  salvar- 
san and  some  three,  and  it  is  considered  that  one  intra- 
muscular injection  of  the  drug  is  equal  to  at  least  three  in- 
travenous injections  so  far  as  the  effect  upon  the  Wasser- 
mann test  is  concerned. 

As  would  be  expected  the  effect  of  treatment  with  sal- 
varsan upon  the  reaction  increases  with  the  number  of 
doses  of  the  drug  that  are  administered,  regardless  of  the 
method  of  administration.  Of  200  cases  receiving  one 
intramuscular  injection  of  salvarsan  of  0.6  gm.,  152,  or 
76  per  cent  became  negative,  while  of  9  cases  receiving 
two  intramuscular  injections,  7,  or  77.7  per  cent  became 
negative.  There  is  little  difference  between  these  two 
groups  of  cases  so  far  as  the  apparent  effect  upon  the 
Wassermann  test  is  concerned,  but  the  number  of  cases 
receiving  two  intramuscular  injections  is  too  small  to  allow 
of  our  basing  any  accurate  statistics  upon,  as  a  larger 


182 


THE   WASSBRMAlSriSr   TEST 


number  of  cases  would  undoubtedly  give  a  higher  per- 
centage in  this  class  of  cases. 

Of  the  cases  treated  by  the  intravenous  method,  177 
were  given  one  intravenous  injection  of  0.6  gm.  of  salvar- 
san,  and  72,  or  40.6  per  cent  became  negative;  52  were 
given  two  intravenous  injections  of  the  same  dose,  of  which 
22,  or  42.3  per  cent  became  negative;  while  10  cases  were 
given  three  intravenous  injections,  of  which  7,  or  70  per 
cent  became  negative.  There  was  but  little  difference  in 
the  percentage  of  negative  results  obtained  in  those  given 
one  and  two  intravenous  injections,  and  it  will  be  noted 
that  two  intravenous  injections  did  not  have  as  much  effect 
upon  the  Wassermann  reaction,  as  one  intramuscular  in- 
jection. The  percentage  of  negative  results  obtained  with 
three  intravenous  injections,  however,  approaches  closely 
to  that  obtained  with  one  intramuscular  injection,  and  jus- 
tifies the  assertion  that  one  intramuscular  injection  is  equal, 
in  its  effect  upon  the  Wassermann  reaction,  to  three  intra- 
venous injections  of  salvarsan. 

The  time  of  the  disappearance  of  a  positive  reaction 
after  treatment  mth  salvarsan  varies  Avith  the  method  of 
administration  as  shown  in  Table  XLII. 

Table  XLII 

The  Eelation  of  the  Method  of  Administration  of  Salvarsan  to  the 

Time  op  Disappearance  of  the  Wassermann  Eeaction  in  Cases 

Treated  with  Salvarsan 


method  op 
administration 

TOTAL 

CASES 

1 

WEEK 

2 

WEEKS 

3 

WEEKS 

4 

WEEKS 

5 

WEEKS 

6 

WEEKS 

7 

WEEKS 

8 

WEEKS 

Intramuscular 

Intravenous 

Combined 

159 
101 

28 

9 
13 

2 

36 

44 
4 

39 

24 

5 

31 

16 

9 

18 
4 
3 

18 

0 
3 

4 

0 

1 

4 
0 
1 

From  the  table  it  will  be  noted  that  the  positive  re- 
action disappeared  more  slowly  after  intramuscular  injec- 
tions of  the  drug  than  after  the  intravenous  injections. 
After  intravenous  injections  there  were  only  four  cases 
that  became  negative  during  the  fifth  week  after  injec- 
tion, and  none  after  that  period,  while  no  less  than  26  cases 


THE   EFFECT   OF   TKEATMENT  183 

treated  by  the  intramuscular  method  became  negative  after 
the  fifth  week.  The  cases  treated  by  the  combined  method 
were  also  slower  in  becoming  negative  than  those  treated 
by  the  intravenous  method  alone. 

If  the  Wassermann  reaction  is  due  to  the  presence  in 
the  tissues  of  living  treponemas,  as  is  now  generally  ac- 
cepted, one  would  expect  that  a  positive  reaction  would 
disappear  more  rapidly  after  an  intravenous  injection  of 
salvarsan  than  after  an  intramuscular  injection.  The  drug, 
when  administered  intravenously,  is  quickly  brought  into 
contact  with  large  numbers  of  the  organisms  and  they  are 
killed,  while  those  not  immediately  affected  and  which  are 
accessible  to  the  drug  perish  later,  but  as  the  drug  is  elim- 
inated within  a  few  da^^s  it  is  obvious  that  what  action  it  has 
occurs  comparatively  quickly  and  if  the  reaction  is  depend- 
ent upon  the  treponemas  and  they  are  destroyed  rapidly, 
the  reaction  also  should  disappear  within  a  comparatively 
short  period  of  time.  On  the  other  hand,  when  salvar- 
san is  administered  intramuscularly  it  is  absorbed  very 
slowly  and  exerts  its  destructive  action  upon  the  trepo- 
nemas for  days  and  weeks,  a  smaller  number  are  killed  in 
a  given  period  of  time,  and  therefore,  the  reaction  persists 
for  a  longer  period  than  after  intravenous  administration. 
Whether  the  positive  Wassermann  reaction  is  due  to  sub- 
stances liberated  from  the  treponema  during  life;  pro- 
duced by  the  tissues  in  answer  to  the  irritation  caused  by 
the  treponema  multiplying  mthin  them;  or  to  substances 
liberated  from  the  organism  after  its  destruction  or  to  the 
reaction  of  the  tissues  to  such  substances,  is  immaterial, 
so  far  as  the  disappearance  of  the  reaction  after  the  admin- 
istration of  salvarsan  is  concerned,  for,  however  produced, 
it  is  evident  that  the  reaction  theoretically  should  dis- 
appear more  rapidly  after  intravenous  injection  than  after 
intramuscular,  and  this  is  what  actually  occurs  in  practice. 

The  Effect  upon  the  Reaction  of  Treatment  with  Mer- 
cury.— The  Wassermann  test  demonstrated  the  true  value 
of  mercury  in  the  treatment  of  syphilis  by  the  fact  that 


184 


THE   WASSERMANN   TEST 


this  drug  is  capable  of  causing  the  disappearance  of  a 
positive  reaction,  but  it  has  also  demonstrated  the  fact  that 
it  is  of  comparatively  small  value  Avhen  compared  with 
salvarsan.  For  one  case  that  becomes  negative  after  treat- 
ment with  mercury  there  are  hundreds  that  become  nega- 
tive after  treatment  with  salvarsan,  and  although  the 
majority  of  both  classes  of  cases  relapse  in  time  and  again 
present  a  positive  Wassermann  reaction,  the  relapses  are 
much  less  frequent  among  patients  treated  with  salvarsan 
than  with  mercury. 

The  relative  efficiency  of  salvarsan  and  mercury  in  caus- 
ing a  disappearance  of  the  Wassermann  reaction  is  well 
illustrated  in  patients  who  had  previously  been  treated 
with  mercury  before  receiving  salvarsan.  Of  90  patients 
who  were  treated  with  mercury  by  the  mouth  for  nine 
months  or  more  before  the  administration  of  salvarsan 
and  who  gave  a  positive  Wassermann  reaction,  68,  or  70.5 
per  cent  became  negative  within  eight  weeks  after  the  ad- 
ministration of  the  drug.  The  intensity  of  the  reaction 
at  the  time  of  receiving  salvarsan  and  the  length  of  time 
the  patients  had  been  taking  mercury  is  given  in  Table 
XLIII. 

Table  XLIII 

The  Wassermann  Test  in  Patients  Who  Had  Taken  Mercury  for 
Various  Periods  of  Time 


METHOD   OF 

TIME   OF 

treatment 

number 

OF  CASES 

CHARACTER   OF   THE 

REACTION 

treatment 

+  + 

+ 

+  - 

Mercury  by  mouth 
(I     '    11        i  I 

11           ii         11 

9  months 

1  year 

2  years 

3  years 

17 
26 
17 

8 

8 

16 

7 

3 

7 
8 
9 
4 

2 
2 
1 

1 

totals 

68 

34 

28 

6 

The  table  shows  that  26  of  these  patients  had  taken  mer- 
cury by  mouth  for  a  period  of  one  year  and  that  16  of 
them  still  gave  a  double-plus  (four-plus)  reaction;  that  17 
had  received  the  same  treatment  for  two  years,  of  whom 
7  still  gave  a  double-plus  reaction ;  and  that  8  had  received 


THE   EFFECT   OF   TREATMENT 


185 


treatment  for  three  years,  of  whom  3  still  gave  a  double- 
plus  reaction.  Of  the  68  cases  not  one  had  become  nega- 
tive as  the  result  of  mercury  administered  by  the  mouth, 
but  after  the  administration  of  salvarsan  every  one  became 
negative  within  8  weeks.  This  is  certainly  very  decisive 
proof  of  the  greater  specific  action  of  salvarsan  and  could 
only  be  ascertained  by  the  effect  of  the  drugs  in  question 
upon  the  Wassermann  reaction. 

Objection  may  be  raised  to  the  results  recorded  above  by 
calling  attention  to  the  fact  that  the  administration  of 
mercury  by  the  mouth  is  now  well  recognized  to  be  the 
very  poorest  of  all  ways  of  giving  this  drug,  although  it 
is  not  so  long  ago  that  three  years '  treatment  by  mouth  with 
mecury  was  considered  by  the  best  authorities  upon  the 
disease  as  adequate  for  the  cure  of  syphilis.  In  answer  to 
this  objection  Table  XLIV  is  given,  covering  18  patients 
treated  with  hypodermic  injections  of  mercury,  a  plan  of 
treatment  generally  acknowledged  to  be  the  most  efficient 
of  the  many  ways  of  administering  the  drug.  For  pur- 
poses of  comparison  the  effect  upon  the  Wasserman  of 
treatment  with  salvarsan  is  also  included. 


Table  XLIV 

The  Comparative  Results  of  Treatment  with  Mercury  and  Salvarsan 
UPON  the  Wassermann  Reaction 


number  of 
cases 

Mermirial  Treatment 

Salvarsan 

Treatment 

injections  of 

GREY  OIL 

CHARACTER    OF 
REACTION 

DOSE   0.6  GM. 

CHARACTER   OF 
REACTION 

2 

7 

+  + 

Intraimiscular 

_ 

1 

8 

+ 

11         ( < 

_ 

1 

9 

+  + 

li         ii 

_ 

1 

11 

+  + 

i  I         11 

_ 

3 

15 

+  + 

Intravenous 

_ 

1 

15 

+ 

Intramuscular 

_ 

4 

18 

+  + 

ii          it 

_ 

2 

19 

+  4- 

Intravenous 

_ 

1 

20 

+  + 

a            a 

_ 

1 

25 

+ 

Intramuscular 

_ 

1 

30 

+ 

( (            a 

- 

From  a  consideration  of  this  table  it  is  evident  that 
cases  having  had  as  many  as  18  to  20  injections  of  grey  oil 


186  THE   WASSERMAIsrE-   TEST 

still  gave  a  double-plus  Wassermann  reaction  and  that 
cases  having  as  high  as  25  and  30  injections  still  gave  a  plus 
(three-plus)  reaction.  After  the  administration  of  salvar- 
san  all  became  negative  within  8  weeks,  and  in  12  of  the 
cases  only  one  intramuscular  injection  of  0.6  gm.  of  sal- 
varsan  was  administered.  While  there  is  reason  to  believe 
that  previous  treatment  with  mercury  may  have  had  some- 
thing to  do  with  the  good  effects  produced  by  salvarsan 
upon  the  reaction,  it  must  be  admitted  that  the  results  ob- 
tained in  these  cases  demonstrate  beyond  all  question  the 
superior  specific  value  of  salvarsan  upon  Treponema  pal- 
lidum. 

Patients  who  have  been  previously  well  treated  with  mer- 
cury show  a  higher  percentage  of  negative  results  after 
treatment  with  salvarsan  than  those  who  have  received  no 
mercurial  treatment.  Thus  of  75  patients  who  had  re- 
ceived mercurial  treatment  before  receiving  salvarsan,  84 
per  cent  became  negative,  while  of  110  patients  who  had 
received  no  treatment  before  the  administration  of  salvar- 
san, only  74.5  per  cent  became  negative. 

In  order  to  illustrate  the  persistence  of  the  positive 
Wassermann  reaction  after  treatment  with  mercurials  Ta- 
ble XLV  has  been  prepared  giving  the  method  of  treat- 
ment, the  length  of  time  the  patient  was  under  treatment, 
the  number  of  cases  observed,  and  the  intensity  of  the 
reaction. 

The  table  illustrates  very  well  the  effect  of  treatment 
with  mercury  upon  the  Wassermann  reaction,  as  it  occurs 
in  the  usual  routine  of  serological  work  in  a  Wassermann 
laboratory.  It  shows  that  continued  treatment  with  the 
drug  for  long  periods  of  time  has  but  little  effect  upon  the 
reaction  in  the  majority  of  instances  and  that  interrupted 
treatment  continued  for  many  years  does  not  produce  a 
negative  reaction  in  many  cases.  It  should  not  be  thought, 
hoAvever,  from  the  data  given  here,  that  the  Wassermann 
reaction  never  becomes  negative  after  treatment  with  mer- 
cury, for  in  a  considerable  proportion  of  cases  it  does,  and 


THE   EFFECT   OF   TKEATMENT 


187 


Table  XLV 

Illustrating  the  Persistence  op  the  Wassermann  Reaction  after 
Treatment  with  Mercury 


method  of 

LENGTH  OF 
TREATMENT 

NUMBER  OF 
CASES 

INTENSITY  OF  THE  REACTION 

TREATMENT 

+  + 

+ 

+  - 

By  mouth 

1  month 

2 

2 

2  months 

3 

1 

2 

3      " 

4 

2 

2 

5      " 

6 

3 

o 

6      " 

10 

7 

1 

2 

7      '' 

4 

3 

1 

8      " 

5 

4 

1 

9      " 

10 

7 

1 

2 

10      '' 

3 

2 

1 

11      '' 

3 

3 

1  year 

24 

13 

6 

5 

14  months 

2 

2 

15      " 

2 

1 

1 

16      " 

4 

3 

1 

17      " 

1 

1 

18      " 

7 

1 

4 

2 

19      " 

4 

3 

1 

20      '' 

2    ■ 

1 

1 

2  years 

15 

8 

5 

2 

2.5  " 

3 

2 

1 

3      " 

5 

1 

2 

2 

4      " 

1 

1 

By  mouth" 

7      " 

1 

1 

By  mouth" 

10      " 

1 

1 

By  mouth* 

12      " 

1 

1 

Inunctions 

2  months 

2 

2 

1 1 

5      " 

1 

1 

Cl 

6      " 

2 

2 

et 

1  year 

1 

1 

^Interrupted  treatment  during  this  time.     +  +  indicates  complete  inhibition  of  hemolysis. 


the  writer  has  observed  many  cases  in  which  a  negative 
Wassermann  reaction  was  obtained  after  from  one  to  two 
years  proper  treatment  with  this  drug  bnt  it  has  been  his 
experience  that  most  cases  becoming  negative  after  mer- 
curial treatment  relapse  in  the  course  of  months  or  a  year 
or  two.  While  he  would  not  go  so  far  as  to  state  that  the 
Wassermann  test  never  becomes  permanently  negative 
after  treatment  with  mercury,  it  has  been  his  experience 
that  it  is  only  in  very  rare  instances  that  a  permanently 
negative  result  is  obtained  from  treatment  with  this  drug 
alone,  no  matter  how  it  is  administered  or  in  what 
dosage.    In  fact,  it  has  been  proved  in  rabbits  experimen- 


188  THE   WASSERMANN   TEST 

tally  infected  with  syphilis,  that  before  the  animals  can  be 
rendered  sterile  as  regards  Treponema  pallidum  so  much 
mercury  has  to  be  administered  as  to  cause  either  the 
death  of  the  animal  or  very  serious  pathological  lesions 
due  to  the  drug.  In  the  writer's  rather  large  experience  he 
has  never  personally  followed  a  case  treated  with  mercury 
alone,  in  which  the  Wassermann  test  was  positive  before 
treatment,  that  became  permanently  negative,  although 
many  cases  have  lost  the  positive  reaction  for  a  while  but 
have  invariably  become  positive  again  upon  the  cessation 
of  treatment.  However,  that  mercury  does  cure  syphilis 
in  some  instances,  is  proved  by  the  fact  that  individuals 
are  encountered  who  undoubtedly  had  the  disease  but  who 
have  been  without  symptoms  for  many  years  and  who  give 
a  negative  "Wassermann  reaction  whenever  tested. 

If  the  patient's  blood  serum  be  carefully  titrated  after 
the  administration  of  either  salvarsan  or  mercury,  it  will 
be  found  that  in  almost  every  instance  the  Wassermann 
reaction  is  influenced  to  some  extent,  although  it  may  never 
become  negative.  Thus,  many  cases  shomng  a  double-plus 
reaction,  or  absolute  inhibition  of  hemolysis,  with  quanti- 
ties of  blood  serum  as  small  as  0.02  c.c.  will  become  nega- 
tive for  this  amount  of  serum,  although  giving  a  positive 
reaction  with  the  usual  quantity  of  serum  used  in  the  rou- 
tine tests ;  i.  e.,  0.1  c.c.  By  titrating  the  inhibitory  strength 
of  each  patient's  serum  it  will  be  found  that,  although  the 
Wassermann  test  may  apparently  be  uninfluenced  when 
the  diagnostic  amount  of  serum  is  used,  that  is,  0.1  c.c, 
some  diminution  in  the  strength  of  the  reaction  will  be 
observed  in  amounts  less  than  0.1  c.c.  In  using  the  Was- 
sermann test  as  a  control  of  treatment  the  titration  of  the 
patient's  serum  is  of  great  importance,  as  will  be  noted. 

N"ot  only  will  treatment  with  salvarsan  or  mercury  mark- 
edly influence  the  strength  of  the  reaction  but  if  treatment 
is  commenced  early  in  the  primary  stage  of  the  disease  it 
will  sometimes  prevent  the  development  of  a  positive  re- 
action, although  clinical  symptoms  may  occur.    The  writer 


WASSERMANN   FiEACTIOlT   AS   CONTROL   OF   TREATMENT     189 

has  observed  several  such  instances,  in  which  secondary 
symptoms  developed  in  spite  of  treatment,  but  in  which  a 
positive  Wassermann  reaction  was  never  obtained. 

The  Wassermann  Reaction  as  a  Control  of  Treatment 

Aside  from  its  value  as  a  diagnostic  agent,  the  great- 
est value  of  the  Wassermann  test  for  syphilis  is  found  in 
its  use  as  a  guide  to  the  treatment  of  the  disease.  It  will 
be  evident,  from  Avhat  has  been  said  of  the  effect  of  treat- ' 
ment  upon  the  positive  reaction  that,  if  scientifically  used, 
the  efficacy  of  any  form  of  treatment  advocated  for  syph- 
ilis may  be  absolutely  determined  by  employing  the  test 
as  a  control  of  that  treatment.  In  the  army  the  test  has 
been  of  the  greatest  practical  value  in  ascertaining  the  rel- 
ative efficiency  of  the  various  methods  of  treating  syph- 
ilis and  it  has  been  very  largely  used  for  this  purpose.  As 
an  illustration  of  the  extent  of  the  use  of  the  reaction  in 
the  army  in  the  control  of  the  treatment  of  S3^philis,  the 
writer  may  say  that  he  has  personally  made  almost  twenty 
thousand  Wassermann  tests  upon  treated  patients,  all  of 
these  tests  being  for  the  purpose  of  controlling  the  treat- 
ment of  the  disease,  and  his  experience  has  been  that  of  all 
other  medical  officers  v/ho  have  had  charge  of  the  army 
laboratories  where  the  Wassermann  test  is  performed. 

By  means  of  the  Wassermann  test,  repeated  at  frequent 
intervals,  one  is  able  to  diagnose  a  relapse  of  syphilitic 
infection  long  before  the  appearance  of  clinical  symptoms, 
and  in  this  way  enabled  to  treat  the  infection  before  it  has 
resulted  in  such  symptoms,  thus  saving  the  patient  much 
anxiety  and  discomfort,  and  obviously  at  a  stage  when  it  is 
much  more  amenable  to  treatment  than  after  gross  clinical 
symptoms  of  relapse  have  occurred.  The  writer  believes 
that  the  first  symptom  of  a  relapse  of  a  syphilitic  infection, 
in  the  vast  majority  of  cases,  is  the  recurrence  of  a  posi- 
tive Wassermann  reaction,  and  that  this  recurrence  often 
happens  weeks  and  even  months  before  marked  clinical 


190  THE   WASSERMANN   TEST 

symptoms  are  noted.  In  other  words,  the  positive  Wasser- 
mann  reaction  is  the  most  delicate  symptom  of  relapse  in 
syphilis  that  is  known,  a  symptom  that  occurs  long  before 
definite  clinical  s^nnptoms,  and  thus  it  is  of  the  greatest 
value  as  an  indication  of  the  need  of  further  treatment  in 
cases  where  it  has  once  disappeared  and  then  recurs.    For 

/this  reason  all  cases  of  syphilis  that  have  been  given  specific 
treatment  should  be  tested  at  frequent  intervals  in  order 
to  ascertain  the  effect  of  the  treatment  upon  the  reaction, 
and  after  a  negative  reaction  is  once  obtained  a  test  should 
be  made  at  least  every  three  months  for  a  period  of  at  least 
one  year,  when,  if  the  reaction  is  still  negative,  a  provoca- 
tive Wassermann  test,  a  Wassermann  test  upon  the  spinal 
fluid,  and  a  luetin  test  should  be  made,  before  the  patient  is 
pronounced  cured. 

The  writer  has  already  mentioned  the  great  importance 
/  of  titrating  the  complement-binding  power  of  the  syphilitic 
1^  patient's  blood  serum  in  judging  of  the  effect  of  any  treat- 
ment upon  the  Wassermann  reaction  and  in  using  the  test 
\?  as  a  control  of  treatment,  and  the  method  of  making  such 
titrations.  The  titration  of  the  patient's  blood  serum  should 
be  done  whenever  it  is  possible,  but  especially  in  those  cases 
that  show  a  continued  double-plus  (four-plus)  reaction 
after  specific  treatment  has  been  administered  for  some 
time.  In  such  cases,  also,  the  spinal  fluid  should  be  tested 
if  the  "Wassermann  on  the  blood  shows  no  diminution  when 
the  serum  is  titrated,  for  it  is  in  this  class  of  cases  that  early 
involvement  of  the  central  nervous  system  is  most  apt  to 
have  occurred. 

The  data  derived  from  the  use  of  the  Wassermann  test 
as  a  control  of  treatment  has  enabled  us  to  use  both  mer- 
cury and  salvarsan  intelligently  and  has  added  greatly 
to  our  knowledge  of  the  action  of  other  drugs  that  have 
been  advocated  from  time  to  time  in  the  treatment  of  the 
disease.  It  has  demonstrated  beyond  all  doubt  that  the 
absence  of  clinical  symptoms  is  no  proof  of  cure  in  this 


WASSERMANlsT   REACTION   AS   CONTROL   OF   TREATMENT     191 

disease,  even  though  symptoms  may  be  absent  for  months 
or  years,  and  that  a  single  negative  Wassermann  reaction 
is  of  absolutely  no  value  in  determining  either  the  question 
of  the  existence  of  syphilis  or  of  its  cure. 

Perhaps  the  most  striking  proof  of  the  value  of  the  Was- 
sermann test  in  the  control  of  the  treatment  of  syphilis  is 
the  results  that  have  been  obtained  by  numerous  workers 
upon  the  occurrence  of  relapse  after  treatment  with  sal- 
varsan,  as  shown  by  the  Wassermann  test.  In  1913,  the 
writer  published""  certain  observations  upon  relapse  in 
syphilis  after  treatment  with  salvarsan  and  has  continued 
collecting  data  upon  this  subject  which  have  confirmed  the 
observations  published.  They  were,  undertaken  for  the 
purpose  of  determining  the  relation  of  relapse  to  the  dose 
and  method  of  administering  the  drug  but  serve  equally 
well  to  illustrate  the  value  of  the  Wassermann  test  in  con- 
trolling the  treatment  of  syphilis  and  in  ascertaining  the 
therapeutic  results  following  treatment  with  a  specific 
drug.  Used  in  this  manner  the  Wassermann  test  has  fur- 
nished the  following  information  regarding  the  occurrence 
of  relapses  after  treatment  with  salvarsan: 

Four  hundred  cases  were  selected  for  study,  and  were 
observed  for  a  period  of  eight  months  or  more.  Of  the  400 
cases,  202,  or  50.5  per  cent  remained  positive  after  the 
administration  of  the  drug,  while  198,  or  49.5  per  cent  be- 
came negative  to  the  Wassermann  test  after  salvarsan  was 
given.  Of  the  198  cases  that  became  negative,  172,  or  86.8 
per  cent  have  relapsed ;  i.  e.,  the  Wassermann  test  has 
again  become  positive. 

The  Stage  of  Syphilis  in  Relation  to  Relapse. — The  re- 
lation of  the  stage  of  syphilis  to  relapse  following  treat- 
ment with  salvarsan  is  shown  in  Table  XL VI. 

A  consideration  of  the  table  shows  that  88.5  per  cent  of 
the  primary  cases  relapsed,  as  shown  by  the  Wassermann 
test ;  84.3  per  cent  of  the  secondary  cases ;  88.8  per  cent  of 
the  latent ;  and  only  69.5  of  the  tertiary  cases.  The  latter 
had  all  received  considerable  treatment  mth  mercury  prior 


192 


THE   WASSERMANN   TEST 


Table  XLVT 

S 

The  Stage  of  Syphilis  in  Relation  to  Relapse  after  Treatment 
WITH  Salvarsan 


stage  of  disease 

NUMBER  op   cases 

NUMBER    OF 
RELAPSES 

%    OF   RELAPSES 

Primary 
Secondary 
Tertiary 
Latent 

35 

115 

23 

25 

31 
97 
16 
22 

88.5 
84.3 
69.5 

88.8 

to  the  administration  of  salvarsan,  so  that  the  compara- 
tively low  percentage  of  relapses  in  this  stage  undoubtedly 
was  due  to  this  fact,  for  the  writer  believes  that  in  un- 
treated cases  the  tertiary  stage  would  show  as  large,  and 
probably  a  larger,  percentage  of  relapses  than  either  the 
primary  or  secondary.  The  latent  stage  showed  the 
largest  percentage  of  relapses  and  this  was  expected,  as 
it  is  this  class  of  cases  that  most  often  prove  resistant  to 
treatment,  so  far  as  the  disappearance  of  the  Wassermann 
test  is  concerned,  but  the  difference  is  only  slight,  as  shown 
by  the  table. 

The  Relation  of  the  Intensity  of  the  Wassermann  Reac- 
tion to  Relapse. — The  cases  giving  the  strongest  Wasser- 
mann reaction  furnished  the  largest  percentage  of  re- 
lapses, as  is  sho\\m  in  Table  XL VII.  Here,  as  in  all  tables 
in  this  work,  the  sign,  ++,  indicates  complete  inhibition  of 
hemolysis,  the  four-plus  reaction  of  many  authorities : 


Table  XLVII 


The  Relation  of  the  Intensity  of  the  Wassermann  Reaction  to  Relapses 
AFTER  Treatment  with  Salvarsan 

character  of 
reaction 

NUMBER  OF  CASES 

NUMBER  OF 
RELAPSES 

%    OF   RELAPSES 

+  4- 

+ 

110 

88 

104 
68 

94.5 

77.2 

198 

172 

86.8 

This  table  demonstrates  that  of  the  110  cases  giving  a 
double-plus  reaction,  no  less  than  104,  or  94.5  per  cent 
relapsed,  while  of  the  88  cases  giving  a  plus  (three-plus 


WASSEEMAI^lil^   REACTIOlSr   AS   CONTROL   OF   TREATMENT     193 

or  two-plus)  reaction,  only  68  or  77  per  cent  relapsed. 
Therefore  it  is  evident  that  the  stronger  the  Wassermann 
reaction  the  greater  the  number  of  relapses  following 
treatment  with  salvarsan. 

Time  of  Relapse  after  Treatment  with  Salvarsan. — By 
controlling  the  treatment  of  syphilis  with  salvarsan  it  has 
been  possible  for  us  to  obtain  accurate  data  regarding  the 
time  of  relapse  after  treatment  with  salvarsan;  i.e.,  the 
period  elapsing  from  the  time  the  Wassermann  test  be- 
came negative  to  the  time  in  which  the  test  again  became 
positive.  The  data  accumulated  show  that,  in  the  major- 
ity of  instances,  a  Wassermann  relapse,  as  it  may  be  called, 
occurs  within  eight  months  after  cessation  of  treatment. 
Thus,  of  the  172  cases  that  relapsed  after  treatment  with 
this  drug,  no  less  than  159  cases  relapsed  within  eight 
months  after  treatment.  Table  XL VIII  gives  the  time  of 
relapse  after  treatment  observed  in  these  cases. 


Table  XLVIII 


The  Time  of  Relapse  after  Treatment  with  Salvarsan 

AS  Shown  ry  the 

Wassermann  Test 

time  op  relapse 

number  of  cases 

time  of  relapse 

number  op  cases 

1  month 

8 

5  months 

17 

5  weeks 

7 

6       " 

12 

6     '' 

5 

7       " 

5 

7     " 

6 

8       '' 

5 

2  months 

38 

9       " 

2 

9  weeks 

1 

10       " 

2 

10     " 

7 

11       " 

1 

11     '' 

2 

12       " 

2 

3  months 

26 

13       '' 

1 

14  weeks 

1 

15       " 

1 

4  months 

19 

22       '' 

1 

24       " 

1 

From  this  table  it  will  be  noted  that  no  less  than  38  cases 
relapsed  at  the  end  of  two  months  and  26  at  the  end  of 
three  months,  so  that  it  would  appear  that  these  periods 
are  of  special  significance  as  regards  the  time  of  relapse. 
It  is  also  interesting  to  note  that  no  less  than  100  of  the 
172  cases  that  relapsed  did  so  by  the  end  of  three  months 
and  no  less  than  149  of  the  172  cases  by  the  end  of  six 


194  THE   WASSEEMAFN   TEST 

months  after  cessation  of  treatment.  From  this  it  would 
appear  that  the  vast  majority  of  cases  of  syphilis  treated 
as  these  were,  with  salvarsan,  will  relapse  within  six 
months  so  far  as  the  Wassermann  reaction  is  concerned, 
and  that  it  is  during  this  period  that  repeated  Wassermann 
tests  are  most  essential. 

The  statement  that  has  already  been  made  regarding  the 
worthlessness  of  a  single  negative  Wassermann  reaction 
is  well  illustrated  by  this  table,  and  even  in  those  cases  in 
which  the  reaction  remains  negative  for  a  long  period  of 
time,  no  assurance  can  be  given  that  it  mil  not  again  be- 
come positive,  for  it  will  be  observed  that  some  of  these 
cases  had  a  negative  Wassermann  reaction  for  as  long  as 
one  and  two  years  and  became  positive. 

As  regards  the  time  of  relapse  in  the  various  stages  of 
syphilis  it  may  be  stated  that  the  Wassermann  tests 
showed  that  the  average  time  of  relapse  in  primary  cases 
was  4^  months;  in  the  secondary  cases,  4  months;  in  the 
tertiary  cases,  3  months;  and  in  the  latent  cases,  31/^ 
months.  The  latent  cases  included  both  early  and  late 
latent  cases,  which  accounts  for  the  longer  period  of  re- 
lapse in  this  class.  As  will  be  noted,  relapses  occur  earlier 
the  longer  the  s^^Dhilitic  infection  has  lasted. 

The  Relation  of  the  Method  of  Administration  of  Salvar- 
san  to  Relapses. — The  Wassermann  test  has  shown  that  the 
percentage  of  relapses  after  salvarsan  varies  considerably 
with  the  method  of  administration  of  the  drug.  The  three 
methods  that  have  been  tested  in  this  way  have  been  the 
intramuscular  injection  of  the  drug,  the  intravenous  in- 
jection, and  the  combined  intramuscular  and  intravenous 
injection.  Table  XLIX  gives  the  number  of  cases  treated 
which  have  been  thus  tested  and  the  number  of  relapses, 
together  with  the  percentage  of  relapse  in  each  class  of 
cases. 

The  number  of  cases  tested  that  were  treated  by  the  com- 
bined intramuscular  and  intravenous  method  was  so  small 
that  the  percentage  of  relapses ;  i.  e.,  66.6  per  cent,  can  not 


WASSERMANN   REACTION   AS   CONTROL   OF   TREATMENT     195 

Table  XLIX 

The  Eelation  of  the  Method  of  Administration  of  Salvarsan  to  Relapse, 
AS  Shown  by  the  Wassermann  Test 


method  of 

total  number 

OF   cases 

relapses 

administration 

NUMBER 

% 

Intramuscular 
Intravenous 

Combined  intramuscular 
and  intravenous 

74 
115 

9 

54 
106 

6 

72.9 
92.1 

66.6 

be  considered  as  a  fair  estimate  of  the  percentage  that 
would  relapse  were  a  greater  number  of  cases  tested. 
However,  the  number  of  cases  tested  that  were  given  sal- 
varsan intramuscularly  and  intravenously  is  large  enough 
to  base  fair  estimates  upon,  and  it  is  very  evident  that  a 
much  smaller  number  of  relapses  follow  the  intramuscular 
method  of  administering  the  drug  than  the  intravenous 
method.  In  justice  to  the  intravenous  method  it  should  be 
stated  that  fully  50  per  cent  of  the  cases  tested  had  only 
received  one  dose  of  the  drug,  but  even  after  two  injections 
the  results  were  not  as  good  as  after  one  intramuscular 
injection.  Of  80  cases  receiving  one  intravenous  injection 
of  0.06  gm.  of  salvarsan  78  relapsed,  or  97.5  per  cent,  while 
of  67  cases  receiving  the  same  dose  intramuscularly,  51 
relapsed,  or  76  per  cent. 

From  what  has  been  stated  regarding  the  study  of  re- 
lapse in  syphilis  after  the  administration  of  salvarsan 
some  idea  may  be  gained  regarding  the  great  value  of  the 
Wassermann  test  when  it  is  used  as  a  control  of  treatment 
or  as  a  test  of  the  efficiency  of  any  drug  recommended  in 
the  treatment  of  the  disease.  The  results  that  have  fol- 
lowed the  modern  method  of  treatment ;  i.  e.,  numerous 
intravenous  injections  of  salvarsan  combined  with  mercu- 
rials, given  in  the  proper  manner,  are  far  superior  to  those 
which  have  been  noted  in  this  chapter  mth  either  drug 
alone.  There  is  but  one  way  of  determining  what  these 
results  are  and  that  is  by  the  use  of  the  Wassermann  test 
as  a  control  of  the  treatment  of  the  disease.  Without  it, 
the  physician  is  at  as  great  a  disadvantage  in  judging  of 


196  THE   WASSEEMAKlSr   TEST 

the  results  of  his  treatment  of  syphilis  as  the  mariner 
would  be  without  a  compass,  and  this  test  may  well  be 
called  the  compass  of  the  physician  in  the  treatment  of 
this  disease. 

The  Interpretation  of  the  Reaction  when  the  Test  is  used 
in  the  Control  of  Treatment. — A  Wassermann  test  should 
not  be  performed  for  the  purpose  of  controlling  the  treat- 
ment of  syphilis  until  treatment  with  salvarsan,  mercu- 
rials, or  other  agents  has  been  omitted  for  at  least  a  month 
and  the  test  should  be  repeated  at  monthly  intervals  re- 
gardless of  the  reaction  if  treatment  is  not  again  instituted. 
If  the  reaction  is  negative  monthly  tests  should  be  made 
in  all  cases  possible.  If  it  remains  persistently  negative 
treatment  should  not  be  persisted  in,  but  if  there  is  any 
trace  of  inhibition  beyond  a  weak  plus-minus  reaction,  the 
treatment  should  be  continued.  It  should  always  he  re- 
membered that  a  single  negative  reaction  is  absolutely  val- 
uless  as  indicating  the  cure  of  the  infection  and  repeated 
tests  must  be  made  with  a  similar  result  before  any  opin- 
ion favoring  cure  is  warranted.  Too  many  patients  have 
been  assured  that  they  were  cured  of  syphilis  on  the 
strength  of  one  or  two  negative  Wassermann  reactions, 
with  disastrous  results  to  innocent  individuals,  as  well  as 
to  the  patient,  and  the  utmost  caution  should  be  exercised 
in  expressing  any  opinion  regarding  ultimate  cure  in  syph- 
ilis. It  should  be  remembered  that  the  Wassermann  test 
has  sho^m  that  the  vast  majority  of  treated  syphilitic  in- 
fections eventually  relapse,  even  though  the  treatment  has 
been  scientific  and  thorough,  so  that  the  prognosis  in  every 
case  should  be  very  guarded. 

In  primary  infections  which  have  been  treated  and  in 
which  the  Wassermann  test  has  become  negative,  a  return 
of  the  reaction  even  to  the  extent  of  a  plus-minus  reaction, 
when  it  is  persistent,  should  indicate  further  treatment. 
In  the  secondary  cases  a  plus-minus  reaction  is  not  of  as 
much  importance,  as  most  of  these  cases  will  give  a 
stronger  reaction  if  they  relapse,  but  even  in  such  cases 


PROVOCATIVE   WASSERMAlSrisr   REACTION  197 

if  the  plus-minus  reaction  persists  for  several  weeks,  treat- 
ment should  be  instituted.  In  tertiary  and  latent  cases  the 
occurrence  of  a  plus  (two-  or  three-plus  of  some  authors) 
reaction  after  treatment  has  been  stopped  should  be  fol- 
lowed by  the  resumption  of  the  treatment. 

Treatment  in  syphilis  should  never  be  discontinued  until 
the  Wassermann  test  has  become  negative  unless  it  is 
found  that  the  case  is  "Wassermann  fast";  i.e.,  that  no 
amount  of  treatment  will  cause  the  reaction  to  become  neg- 
ative. Wassermann  fast  cases  are  frequently  met  with  in 
practice  and  occur  during  every  stage  of  the  disease  but 
such  cases  occur  more  frequently  in  tertiary  and  latent 
infections  than  in  primary  or  secondary.  In  the  "Was- 
sermann fast"  cases  the  clinical  symptoms,  aside  from  the 
positive  Wassermann  reaction,  may,  and  generally  do,  dis- 
appear under  treatment,  but  the  reaction  never  becomes 
negative. 

The  Provocative  Wassermann  Reaction 

In  1910,  Gennerich^°^  called  attention  to  the  fact  that 
cases  presenting  a  negative  Wassermann  reaction  often 
showed  a  positive  reaction  after  the  administration  of  a 
dose  of  salvarsan,  this  reaction  occurring  either  within  a 
few  hours  after  the  injection  or  within  two  weeks  follow- 
ing it.  He  called  attention  to  the  value  of  this  phenomenon 
in  the  diagnosis  of  obscure  syphilitic  infections,  and  as  a 
method  of  determining  whether  a  patient  is  really  cured 
of  the  infection.  He  suggested  that  this  so-called  "pro- 
vocative Wassermann"  be  used  in  treated  cases  that  had 
presented  negative  reactions  for  some  time,  and  were  pre- 
sumably cured,  as,  in  such  instances  he  had  found  the 
provocative  test  frequently  positive,  thus  demonstrating 
that  the  disease  was  still  present.  Milian,"^  of  Paris, 
about  the  same  time,  called  attention  to  the  value  of  the 
procedure  in  the  study  of  the  disease,  as  well  as  Herx- 
heimer,^°®  and  Michaelis,^"  but  to  Gennerich  belongs  the 


198  THE   WASSERMAITN   TEST 

credit  of  first  actually  applying  the  test  in  the  diagnosis 
of  syphilis  and  as  a  control  of  the  treatment  of  the  disease. 

Method  of  Making  a  Provocative  Wassermann  Test. — 
The  writer  recommends  the  following  procedure  in  making 
a  provocative  Wassermann  test:  The  suspected  patient 
is  given  from  0.3  to  0.6  gm.  of  salvarsan  intravenously  and 
the  blood  is  collected  and  tested  12  hours  after  the  admin- 
istration of  the  drug;  24  hours  after  administration;  and 
every  day  for  at  least  ten  days.  If  the  negative  reaction 
is  to  become  positive,  it  generally  does  so  within  from  a 
few  hours  to  5  or  6  days  after  the  salvarsan  has  been 
given,  but  delayed  reactions  have  occurred  not  infre- 
quently up  to  10  days  after  administration,  and  it  is  good 
practice  to  make  a  test  upon  the  12th  and  14th  days  after 
administration,  in  addition  to  those  recommended.  Dur- 
ing the  first  two  days  after  salvarsan  has  been  given,  if 
it  is  possible,  the  blood  should  be  collected  at  intervals  of 
three  or  four  hours  and  tested,  as  sometimes  the  reaction 
is  very  evanescent  in  character  and  quickly  disappears. 
However,  owing  to  the  labor  involved  in  making  so  many 
tests  it  is  seldom  practicable  to  do  this,  and  the  procedure 
first  mentioned  will  generally  prove  sufficient  to  detect  the 
reaction,  if  it  occurs.  Stokes  and  O'Leary^^^  recoromend 
that  the  blood  be  dra^wm  at  the  time  of  injection  and  daily 
thereafter  for  one  week,  and  that  all  blood  collected  be 
tested  with  several  antigens  at  one  sitting  and  with  the 
same  reagents,  in  order  to  avoid  any  difference  in  the  re- 
sults that  might  occur  from  variations  in  the  reagents  used 
in  the  test  from  day  to  day. 

Results  of  the  Test. — In  a  recent  contribution,  King,"^  as 
the  result  of  his  investigation  of  the  provocative  test  upon 
a  very  small  number  of  patients,  came  to  the  conclusion 
that  the  provocative  Wassermann  reaction  does  not  exist, 
but  that  the  positive  result  that  sometimes  follows  an  in- 
jection of  salvarsan  is  due  either  to  normal  variations  in 
the  complement-binding  power  of  s^^philitic  serum,  as 
shown  by  the  writer,  or  to  variations  in  the  technic  or 


PKOVOCATIVE   WASSERMAISTN   REACTIOlSr 


199 


reagents  used  in  the  test  when  made  upon  different  days. 
His  work  is  very  far  from  convincing  and  the  united  ex- 
perience of  hundreds  of  observers  who  have  used  the  pro- 
vocative test  in  their  laboratories  and  practice  is  unan- 
imous as  regards  the  actual  occurrence  of  this  phenom- 
enon and  as  to  the  value  of  the  test  in  the  diagnosis  of 
syphilis  and  the  control  of  its  treatment. 

Nichols,^^^  after  stating  that  it  is  a  well-established  fact 
that  in  a  syphilitic  a  negative  Wassermann  reaction  can  be 
converted  into  a  positive  one  by  an  injection  of  salvarsan, 
gives  the  following  table  illustrative  of  the  effect  of  the 
provocative  test  in  ten  patients  in  whom  a  luetin  test  was 
also  performed  at  the  same  time.  Table  L  gives  the  re- 
sults that  he  obtained  in  these  cases : 

Table  L 

Eesults  of  the  Provocative  Wassermann  Test  and  the  Luetin  Test. 
(After  Nichols.) 


TIME  ORDINARY  WASSERMANN 

PROVOCATIVE 

CASE   NO. 

REACTION   HAD  BEEN 

LUETIN  TEST 

WASSERMANN 

NEGATIVE 

TEST 

1 

16  months 

_ 

_ 

2 

21       '' 

_ 

_ 

3 

24       '' 

_ 

_ 

4 

24       " 

_ 

_ 

5 

24       '' 

_ 

_ 

6 

15       '' 

+ 

+  4- 

7 

17       " 

+ 

+  + 

8 

18       " 

+ 

+  + 

9 

19       '' 

+ 

+  4- 

10 

? 

+ 

+  + 

This  table  is  conclusive  proof  of  the  occurrence  of  the 
provocative  reaction,  as  it  shows  complete  agreement  be- 
tween the  results  of  this  test  and  the  luetin  test,  for  it  is 
hardly  probable  that,  if  the  positive  reaction  in  these  cases 
was  due  to  variation  in  the  complement-binding  power  of 
the  blood  serum  or  inaccuracies  in  technic,  the  results  of 
the  provocative  test  would  be  identical  with  the  results  of 
the  luetin  test  in  every  case. 

Nichols  calls  attention  to  Case  10,  in  the  above  table,  be- 


200  THE   WASSERMANN"   TEST 

cause  of  the  interesting  results  obtained  as  regards  a 
delayed  provocative  reaction.  The  patient  was  infected 
in  1906  and  had  two  years  of  treatment  by  the  mouth  with 
mercury  and  potassium  iodide.  No  symptoms  had  oc- 
curred since  1908  and  in  March,  1913,  he  gave  a  negative 
reaction.  On  April  18th  he  was  given  an  intravenous  injec- 
tion of  0.6  gm.  of  salvarsan  and  his  blood  was  tested  every 
day  with  the  following  results :  April  18th,  negative ;  April 
19th,  negative;  April  20th,  negative;  April  21st,  negative; 
April  22nd,  negative;  April  23rd,  double-plus  (four-plus). 

This  case  well  illustrates  the  value  of  the  provocative 
reaction  in  patients  who  have  had  no  symptoms  of  syphilis 
for  several  years  and  who  give  a  negative  Wassermann  test. 

Stokes  and  O'Leary,"*  in  a  very  excellent  study  of  the 
provocative  Wassermann  reaction,  obtained  very  signifi- 
cant results.  They  made  103  provocative  tests,  of  which 
19,  or  18.4  per  cent  resulted  in  a  reversal  of  the  ordinary 
Wassermann  from  negative  to  positive.  This  small  per- 
centage of  positive  results  with  the  test  is  not  unusual  and 
it  has  been  the  writer's  experience  that  it  is  seldom  that 
a  higher  percentage  than  20  per  cent  is  obtained  with  the 
provocative  Wassermann  test  upon  limited  groups  of 
cases.  If  thousands  of  tests  were  made  it  is  probable  that 
the  positive  percentage  Avould  be  somewhat  higher  but 
from  18  to  20  per  cent  of  positive  results  is  about  all  that 
can  be  expected  from  the  reaction. 

As  to  the  character  of  the  cases  showing  a  positive  re- 
action with  the  test,  Stokes  and  O'Leary  state  that  26.3 
per  cent  presented  no  clinical  symptoms  of  syphilis,  but 
that  the  remainder  show  suspicious  symptoms  although 
the  Wassermann  was  negative.  However,  they  state  that 
''In  no  one  of  the  positive  cases  were  the  signs  so  indu- 
bitable that  the  provocative  could  be  regarded  as  unneces- 
sary or  merely  confirmatory."  They  say  further:  *'we 
believe  the  test  has  distinct  value  in  determining  the  status 
of  a  patent  under  treatment. ' ' 

Stokes  and  O'Leary  give  Table  LI  showing  the  results 


PKOVOCATIVE   WASSERMAI^N   EEACTIOjST 


201 


obtained  with  the  provocative  Wassermann  test  in  differ- 
ent types  of  syphilis. 

Table  LI 
Efficiency  of  the  Provocative  Test  in  Various  Types  op  Syphilis 


TYPE 

NUMBER  OF  CASES 

%   POSITIVE 

Heredosyphilis 

2 

0 

Osseous 

5 

20 

Central  nervous  system 

12 

25 

Vascular 

3 

33 

Latent 

10 

40 

Late  cutaneous 

5 

60 

Late   mucous   membrane 

5 

80 

While  they  admit  that  the  nmnber  of  cases  treated  is 
not  large  enough  to  base  accurate  conclusions  upon,  they 
state  that,  from  their  series,  it  appears  as  though  the  pro- 
vocative test  is  least  useful  in  the  class  of  cases  where  it  is 
most  needed,  but,  even  if  this  proves  to  be  true,  and  we 
can  only  expect  20  per  cent  of  positive  results  in  osseous 
syphilis,  25  per  cent  in  syphilis  of  the  central  nervous 
system,  and  33  per  cent  in  vascular  syphilis,  the  writer 
believes  that  the  test  is  well  worth  while,  as  often  it  will 
be  the  only  method  available  for  making  a  diagnosis  in 
this  class  of  cases. 

The  indications  for  a  provocative  Wassermann  reaction 
are  given  by  Stokes  and  O'Leary  in  their  paper  and  are 
so  admirable  that  this  section  of  the  paper  is  here  quoted : 

"1.  A  definite  history  of  primary  or  secondary  lesions 
or  a  suspicious  genital  sore  of  any  description.  (With  a 
negative  Wassermann.) 

"2.  Syphilis  in  husband  or  wife  or  a  history  of  a  sore 
in  either. 

''3.  Treated  cases  to  determine  the  fact  of  cure  or  need 
for  further  treatment.  One-third  of  the  cases  thus  tested 
by  us,  gave  a  positive  provocative  effect. 

"4.  Obscure  bone  or  joint  lesions. 

"5.  Histories  of  miscarriages  unless  the  anatomical 
cause  is  glaringly  obvious. 


202 


THE   WASSERMANN   TEST 


"6.  Mothers  of  syphilitic  children  without  clinical  signs 
of  the  disease. 

'^7.  Cases  with  a  history  of  a  positive  AVassermann  else- 
where, negative  on  present  examination. 

"8.  Mental  deviates  and  constitutionally  inferior  indi- 
viduals with  suspicious  histories. 

''9.  Certain  signs  elicited  by  special  examinations,  such 
as  decreased  bone  conduction  with  normal  hearing,  chorio- 
retinitis and  retinitis  pigmentosa,  bilateral  dacryocystitis  in 
childhood,"  etc. 

In  all  the  above  conditions  the  writer  agrees  with  the 
authors  that  a  provocative  Wassermann  test  should  be 
made,  if  possible,  for  in  nearly  all  cases  in  which  the  indi- 
cations for  the  test  cited  have  occurred,  the  writer  has 
found  the  test  of  service. 

As  an  illustration  of  the  results  obtained  with  the  test 
in  cases  of  s^^philis  that  have  not  shown  symptoms  for  a 
long  period  of  time  and  in  which  the  ordinary  Wassermann 
test  has  been  negative  for  months  and  even  years.  Table 
LII  is  given : 

Table  LII 
Results  of  Provocative  Wassermann  Test  in  Syphilis 


TIME  SINCE 
TREATMENT 

TIME  SINCE  WAS- 

RESULT OP 

RESULT  OF 

CASE  NO. 

SERMANN  BECAME 

WASSERMANN 

PROVOCATIVE 

NEGATIVE 

REACTION 

REACTION 

1 

12  months 

12  months 

_ 

+  4- 

e 

13  " 

13  " 

_ 

_ 

3 

14   " 

14   " 

_ 

+  + 

4 

15  " 

15  " 

_ 

+  + 

5 

16   " 

16  " 

_ 

_ 

6 

17   '' 

17   " 

_ 

+  + 

7 

18  " 

18  " 

_ 

+  + 

8 

19  " 

19  " 

_ 

+  + 

9 

20   " 

20  " 

_ 

+  + 

10 

21  " 

21  " 

_ 

_ 

11 

24  " 

24   " 

_ 

_ 

12 

24  '' 

24  " 

_ 

+  + 

13 

24  " 

24  " 

_ 

_ 

14 

25  " 

25  '' 

- 

+  + 

The  cases  given  in  the  above  table  are  selected  from  a 
large  number  because  they  show  the  results  of  the  provoc- 


PROVOCATWE    WASSERMAI^F    EEACTION  203 

ative  reaction  month  after  month,  as  regards  time,  and 
because  the  data  relating  to  them  is  of  such  a  nature  as  to 
make  the  test  definite  and  conclusive.  It  will  be  seen  that 
a  positive  reaction  was  obtained  as  long  as  25  months  after 
the  cessation  of  treatment,  during  which  time  no  symptoms 
of  syphilis  had  been  noted  and  the  Wassermann  reaction 
was  always  negative,  when  tested. 

The  writer  is  firmly  convinced,  from  personal  experience, 
that  a  dose  of  salvarsan  or  neosalvarsan  will  frequently 
convert  a  negative  "Wassermann  reaction  into  a  positive 
one  and  that  this  phenomenon  is  not  due  to  any  variation 
occurring  normally  in  the  patient's  blood  serum  or  to  dif- 
ferences in  technic  or  in  the  reagents  used  in  making  the 
test.  When  one  has  tested  cases  week  after  week,  always 
with  a  negative  result,  and  when  these  same  cases  after  a 
dose  of  salvarsan  or  neosalvarsan  become  positive  within 
a  few  hours  or  days  after  the  administration  of  the  drug, 
there  can  be  only  one  conclusion,  and  that  is  that  the  posi- 
tive result  has  been  produced  by  the  drug,  and  the  writer 
has  had  this  experience  too  many  times  to  allow  of  the 
least  doubt  in  his  mind  of  the  existence  of  a  provocative 
reaction  following  the  administration  of  these  drugs.  It 
is  true  that  the  test  often  fails  in  cases  in  which  one  would 
expect  it  to  be  positive,  but  this  is  no  proof  of  the  non- 
existence of  the  phenomenon. 

Cause  of  the  Provocative  Reaction. — The  exact  nature  of 
the  provocative  reaction  is  still  undetermined.  There  are 
two  theories  that  have  been  discussed  in  this  connection, 
the  first  being  that  the  reaction  is  due  to  the  stimulation 
of  the  treponemas  by  the  drug  and  the  consequent  pro- 
duction of  more  of  the  substance  or  substances  which  give 
rise  to  a  positive  Wassermann  reaction;  while  the  second 
is  that  the  drug  kills  sufficient  of  the  treponemas  to  increase 
the  amount  of  toxin  and  thus  the  syphilitic  antibody,  as 
it  may  be  called,  is  increased.  The  first  theory  depends 
upon  the  belief  that  Treponema  pallidum,  during  its  life 
and  multiplication  in  the  tissues  causes  the  production  of 


204  THE   WASSERMANN   TEST 

some  substance  wliich  produces  the  Wasserraann  reaction, 
and  that  a  small  dose  of  salvarsan  stimulates  the  few  re- 
maining organisms  in  the  negative  Wassermann  cases  to 
overproduction  of  this  substance  for  a  short  time,  and  thus 
the  negative  Wassermann  reaction  becomes  positive.  The 
second  theory  rests  upon  the  belief  that  the  organisms 
killed  by  the  drug  either  liberate  the  substance  producing 
the  positive  reaction,  or  a  toxin  which  stimulates  the  pro- 
duction of  this  substance  in  the  tissues  of  the  body.  Neither 
theory  has  been  proved  correct  as  we  know  nothing  cer- 
tain as  to  the  actual  cause  of  the  Wassermann  reaction. 

Some  authorities  have  recommended  intramuscular  in- 
jection of  mercury  for  the  production  of  the  provocative 
Wassermann  reaction  but  the  reports  concerning  the  effi- 
cacy of  this  method  are  so  conflicting  that  it  is  not  recom- 
mended. 

The  provocative  Wassermann  test  is  a  useful  adjunct 
to  our  diagnostic  methods  for  syphilis  and  should  be  re- 
sorted to  whenever  it  is  possible,  both  in  the  diagnosis  of 
the  disease  and  in  determining  the  question  of  the  cure  of 
the  infection.  The  large  amount  of  work  involved  in  mak- 
ing the  numerous  tests  that  are  necessary  in  correctly  us- 
ing the  provocative  reaction  will  always  operate  to  limit 
its  use  to  comparatively  few  cases  but  whenever  it  is  pos- 
sible to  use  the  test  it  should  be  employed. 

From  the  evidence  that  has  accumulated  it  may  be  stated 
that  in  patients  who  have  presented  a  negative  Wasser- 
mann reaction  for  long  periods  of  time  and  in  whom  symp- 
toms of  syphilis  have  heen  absent  during  this  period,  the 
provocative  Wassermann  test  often  results  positively.  The 
same  result  is  sometimes  obtained  in  cases  presenting  sus- 
picious symptoms  but  in  which  the  ordinary  Wassermann 
test  is  negative,  or  in  cases  vjhich  give  a  history  of  infec- 
tion but  with  no  symptoms  and.  a  negative  Wassermann  re- 
action. 


CHAPTER  X 

THE  WASSERMANN  TEST  UPON  THE  CEREBRO- 
SPINAL FLUID.  INTERPRETATION  OF  RE- 
SULTS. THE  COLLOIDAL  GOLD  TEST. 
THE  CELL  COUNT  AND  TESTS  FOR 
INCREASE  IN  GLOBULINS  IN 
THE  CEREBROSPINAL 
FLUID 

Recent  observations  have  shown  the  great  importance 
of  the  Wassermann  test  upon  the  cerebrospinal  fluid  and 
it  may  be  stated  that  no  case  of  syphilis  can  be  considered 
as  having  had  the  best  treatment  unless  this  fluid  has  been 
tested,  even  though  the  Wassermann  test  upon  the  blood 
is  negative  and  the  patient  has  been  free  from  symptoms 
for  long  periods  of  time.  It  is  now  demonstrated  beyond 
doubt  that  involvement  of  the  central  nervous  system  fre- 
quently occurs  very  early  in  this  disease  and  this  demon- 
stration has  been  rendered  possible  by  the  results  of  the 
Wassermann  test  upon  the  cerebrospinal  fluid  during  the 
early  latent  and  secondary  stages  of  the  infection. 

The  Wassermann  test  upon  the  cerebrospinal  fluid  often 
shows  involvement  of  the  central  nervous  system  before  any 
clinical  s;^Tiiptoms  are  observed  and  the  use  of  the  test 
has  rendered  intensive  treatment  possible  and  has  resulted 
in  either  preventing  the  development  of  such  symptoms  or 
in  greatly  delaying  their  appearance.  Formerly  it  was  be- 
lieved that  if  the  test  gave  a  negative  reaction  in  the  cere- 
brospinal fluid  and  a  positive  one  in  the  blood,  the  diag- 
nosis of  cerebrospinal  syphilis  should  be  made,  the  result 
serving  to  distinguish  this  condition  from  paresis,  in  which 
both  the  blood  and  cerebrospinal  fluid  usually  gave  a  posi- 

205 


206  THE    WASSERMANl^    TEST 

tive  reaction.  However,  it  has  since  been  shown  that  the 
negative  result  in  the  cerebrospinal  fluid  depended  en- 
tirely upon  the  amount  of  the  fluid  used  in  the  test 
and  that  if  a  large  enough  amount  was  used  a  positive 
result  could  be  obtained  in  the  fluid  in  almost  100  per  cent 
of  cases  of  cerebrospinal  syphilis.  Therefore,  it  follows, 
that  the  Wassermann  test  upon  the  cerebrospinal  fluid,  so 
far  as  its  value  in  differentiating  various  syphilitic  con- 
ditions of  the  central  nervous  system  is  concerned,  depends 
upon  quantitative  factors  entirely,  so  that  it  is  necessary 
in  testing  this  fluid  to  test  different  quantities  if  one  is  to 
secure  the  best  results  from  the  test.  The  importance  of 
the  Wassermann  test  upon  the  cerebrospinal  fluid  is  shown 
by  the  statement  of  Fordyce"^  that  ''From  25  to  35  per 
cent  of  syphilitic  individuals  have  positive  findings  in  the 
spinal  fluid  during  the  first  year  of  their  infection"  and 
during  this  time  symptoms  referable  to  the  central  nervous 
system  are  frequently  present.  This  being  so,  the  impor- 
tance of  ascertaining  in  every  case  of  syphilis  whether 
the  central  nervous  system  is  involved  at  as  early  a  date 
as  possible,  can  hardly  be  overestimated,  and  for  this  rea- 
son the  cerebrospinal  fluid  should  be  tested  as  well  as  the 
blood  serum,  although  the  necessity  of  the  test  upon  the 
spinal  fluid  has  not,  as  yet,  been  fully  realized  by  the  medi- 
cal profession. 

The  writer  has  already  described  the  method  of  obtain- 
ing the  cerebrospinal  fluid  and  the  technic  required  (Chap- 
ter V)  in  making  the  test.  As  the  differentiation  of  some 
of  the  syphilitic  diseases  of  the  central  nervous  system 
depends  upon  the  quantity  of  fluid  tested,  the  writer  rec- 
ommends that  0.2,  0.5,  0.7  and  1  c.c.  quantities  be  tested 
in  each  case,  with  his  method  of  performing  the  reaction, 
the  fluid,  of  course,  not  being  inactivated  before  use.  The 
quantity  of  spinal  fluid  tested,  with  any  method,  should 
always  be  at  least  four  times  the  quantity  of  blood  serum 
tested  with  the  particular  method  used,  and  it  is  always 
best  to  use  both  smaller  and  larger  amounts  than  this  in 


CEREBROSPINAL   FLUID  207 

order  to  determine  the  strength  of  the  positive  reaction 
upon  which  depends  the  differential  diagnosis  of  the  con- 
dition present. 

Hauptmann  and  Hossli"®  Avere  the  first  to  insist  upon 
the  nse  of  large  quantities  of  the  cerebrospinal  fluid  in  ap- 
plying the  Wassermann  reaction  and  their  work  changed 
entirely  the  prevalent  conception  of  the  comparative  rarity 
of  a  positive  reaction  in  diseases  like  tabes  and  demon- 
strated that  almost  any  syphilitic  disease  of  the  central 
nervous  system  would  give  a  positive  Wassermann  re- 
action with  these  larger  amounts  of  fluid.  Thus,  the  find- 
ing of  a  positive  reaction  in  the  cerebrospinal  fluid,  once 
looked  upon  as  almost  pathognomonic  of  paresis,  has 
ceased  to  point  to  this  disease  unless  it  is  obtained  with 
small  amounts  of  the  fluid,  for  with  large  amounts  other 
syphilitic  diseases  of  the  central  nervous  system  also  give 
a  positive  reaction. 

Because  of  the  use  of  too  small  amounts  of  the  cerebro- 
spinal fluid  in  making  the  Wassermann  test  much  of  our 
statistical  data  regarding  the  occurrence  of  a  positive  re- 
action in  the  various  syphilitic  infections  of  the  central 
nervous  system  are  worthless  and  a  great  many  divergent 
results  are  reported  in  the  literature  owing  to  this  fact. 
Mueller"^  obtained  75  per  cent  of  positive  reactions  in 
tabes,  and  98  per  cent  in  paresis,  while  Boas  found  that 
all  of  his  untreated  cases  of  tabes  gave  a  positive  reaction. 
This  is  a  very  good  illustration  of  the  confusion  in  results 
due  to  the  use  of  different  amounts  of  the  spinal  fluid,  for 
Boas  used  larger  amounts  of  the  fluid  in  his  tabetic  cases 
and  obtained  a  much  higher  percentage  of  positive  results. 

The  writer's  experience  may  be  summed  up  as  follows: 

In  paresis  the  AYassermann  test  is  positive  in  the  blood 
in  nearly  100  per  cent  of  the  cases,  and  if  1  c.c.  of  the 
cerebrospinal  fluid  be  used,  it  is  positive  in  practically 
100  per  cent  of  the  cases.  If  smaller  amounts  of  the  spinal 
fluid  be  tested  the  percentage  of  positive  reactions  will  be 


208  THE   WASSEEMAli5"N   TEST 

fonnd  to  decline,  until  when  but  0.2  c.c.  of  the  fluid  is  tested 
only  from  75  to  80  per  cent  of  the  cases  ^nll  give  a  positive 
reaction.  Allien  0.5  c.c.  of  the  fluid  is  used  practically  98 
per  cent  of  cases  of  paresis  will  give  a  positive  reaction  in 
the  spinal  fluid,  a  result  never  found  in  any  other  syphilitic 
condition  of  the  central  nervous  system. 

In  cerebrospinal  sypliilis,  if  1  c.c.  of  the  spinal  fluid  be 
tested  almost  100  per  cent  of  the  cases  will  prove  positive, 
but  if  only  0.2  c.c.  of  the  fluid  be  tested  only  about  10  per 
cent  will  prove  positive.  The  AVassermann  on  the  blood 
in  these  cases  gives  from  60  to  75  per  cent  of  positive  re- 
sults, as  reported  by  different  investigators. 

In  tabes,  if  1  c.c.  of  the  cerebrospinal  fluid  be  tested,  the 
Wassermann  will  be  positive  in  about  95  per  cent  of  the 
cases,  but  if  only  0.2  c.c.  of  the  fluid  be  tested  the  positive 
results  will  only  amount  to  from  5  to  10  per  cent.  In  this 
disease  the  Wassermann  test  upon  the  blood  gives  about 
70  per  cent  of  positive  reactions. 

From  the  above  it  is  apparent  that  in  differentiating  be- 
tween paresis,  cerebrospinal  syphilis  and  tabes,  the  ac- 
curate titration  of  the  complement-binding  power  of  the 
cerebrospinal  fluid  is  most  important.  If  1  c.c.  of  the  fluid 
is  used  for  the  test,  the  results  mil  be  positive  in  prac- 
tically all  of  the  cases  of  either  disease,  but  if  smaller 
amounts  of  the  fluid  be  used  it  will  be  found  that  results 
are  obtained,  which,  if  taken  in  conjunction  with  the  clin- 
ical sjrmptoms  present,  will  generally  enable  the  correct 
diagnosis  to  be  made.  Thus,  if  a  positive  reaction  is  ob- 
tained with  0.2  c.c.  of  the  fluid,  or  less,  the  diagnosis  is  al- 
most certainly  paresis,  for  only  from  5  to  10  per  cent  of 
cases  of  cerebrospinal  sj^hilis  or  tabes  give  a  positive 
result  Avith  this  amount  of  fluid.  If  a  positive  reaction 
is  obtained  with  0.1  c.c.  of  the  fluid  the  diagnosis  of  paresis 
will  be  found  correct  in  99  per  cent  of  the  cases  examined. 

The  Test  upon  the  Cerebrospinal  Fluid  in  the  Various 
Stages   of   Syphilis. — As   stated,   recent   researches   have 


CEREBROSPINAL   FLUID  209 

shown  that  involvement  of  the  central  nervous  system  in 
syphilis  occurs  early  in  the  disease  in  a  considerable  pro- 
portion of  the  cases,  as  has  been  shown  by  the  results  of 
the  Wassermann  test  upon  the  cerebrospinal  fluid.  In  fact, 
so  good  an  authority  as  Fordyce  holds  that  paresis,  tabes, 
and  the  majority  of  other  manifestations  of  syphilis  on 
the  part  of  the  nervous  system  originate  ''at  the  time  of 
the  general  infection  in  the  first  year"  and  there  is  much 
to  support  this  theory  in  the  biological  and  clinical  char- 
acteristics of  syphilitic  infection. 

Results  in  Primary  Stage. — The  writer  has  no  knowledge 
of  a  well  authenticated  positive  result  of  the  Wassermann 
test  upon  the  cerebrospinal  fluid  in  the  primary  stage  of 
syphilis. 

Results  in  the  Secondary  Stage. — The  older  writers  upon 
this  subject  all  agreed  in  stating  that  cases  showing  no 
symptoms  of  involvement  of  the  central  nervous  system,  in 
the  secondary  stage  of  s^^hilis,  were  invariably  negative 
to  the  Wassermann  test  upon  the  spinal  fluid,  or  to  other 
tests  upon  this  fluid,  while,  if  symptoms  of  involvement  were 
present  the  Wassermann  was  generally  positive.  One  ex- 
ception may  be  made  to  this  statement  in  the  case  of 
Eavaut"^  who,  in  1903,  reported  that  among  116  cases  in 
which  the  spinal  fluid  was  tested,  no  less  than  67  per  cent 
showed  some  abnormality  of  the  fluid,  either  in  an  in- 
creased cell  count  or  an  increase  in  globulins.  The  more 
recent  investigators  have  reported  numerous  instances  in 
which  the  Wassermann  test  upon  the  cerebrospinal  fluid 
was  positive  early  in  the  secondary  stage,  even  when  there 
were  no  symptoms  of  involvement  of  the  central  nervous 
system.  Thus  Wile^^°  found  30  per  cent  of  the  cases  of 
secondary  syphilis  that  he  tested  gave  a  positive  result  with 
the  Wassermann  test  upon  the  spinal  fluid,  and  Craig  and 
Collins^^^  found  that  a  considerable  proportion  of  their 
early  secondary  cases  gave  a  positive  Wassermann  reac- 
tion in  the  spinal  fluid.     Ellis  and  Swift^^^  examined  22 


210  THE   WASSEEMAFN   TEST 

cases  of  untreated  early  secondary  syphilis  and  found  the 
spinal  fluid  negative  in  all,  but  in  56  cases  of  late  secondary 
syphilis,  tested  by  Altman  and  Dreyfus,^^^  no  less  than 
eleven  gave  a  positive  Wassermann  reaction  in  the  spinal 
fluid. 

Results  in  the  Tertiary  St£ige. — The  results  of  the  Was- 
sermann test  upon  the  cerebrospinal  fluid  in  paresis,  cere- 
brospinal syphilis,  and  tabes  have  already  been  discussed, 
but  it  should  be  remembered  that  the  examination  of  the 
fluid  in  this  stage  of  the  disease  from  many  cases  in  which 
there  are  no  discoverable  lesions  in  the  central  nervous 
system  will  result  positivel)^,  as  stated  by  Kolmer.^^*  The 
writer  has  repeatedly  observed  cases  of  tertiary  syphilis 
that  gave  a  positive  Wassermann  reaction  with  the  cere- 
brospinal fluid  but  in  which  there  was  absolutely  no  evi- 
dence of  involvement  of  the  central  nervous  system,  but 
he  would  be  loath  to  conclude  from  this  that  no  lesions  due 
to  Treponema  pallidum  were  present.  At  any  rate  such 
a  finding  is  the  signal  for  a  very  careful  scrutiny  of  the 
patient  and  a  very  guarded  prognosis  as  regards  future 
involvement  of  the  central  nervous  system. 

Results  in  the  Latent  Stage. — The  results  of  the  test 
upon  the  cerebrospinal  fluid  in  the  latent  stage  of  syphilis 
mil  vary,  of  course,  with  the  involvement  of  the  central 
nervous  system,  but  the  percentage  reported  by  most  ob- 
servers does  not  exceed  15  per  cent,  including  both  early 
and  late  latent  infections. 

The  Interpretation  of  the  Results  of  the  Test 

The  interpretation  of  the  results  of  the  Wassermann 
test  upon  the  cerebrospinal  fluid,  as  regards  the  mtensity 
of  the  reaction,  are  the  same  as  already  noted  in  the  dis- 
cussion of  the  interpretation  of  the  results  upon  the  blood, 
with  this  exception;  that  a  positive  Wassermann  reaction 
upon  the  cerebrospinal  fluid  is  specific  of  syphilis,  in  the 
opinion  of  practically  all  observers.    The  v/riter  has  never 


CEREBROSPINAL   ELUID  211 

seen  a  positive  reaction  upon  the  spinal  fluid  in  any  other 
condition  than  syphilis  and  his  experience  is  that  of  all  who 
have  studied  this  phase  of  the  subject. 

It  is  also  true  that  more  double-plus  (four-plus)  reac- 
tions are  observed  in  the  spinal  fluid  than  in  the  blood 
although  occasionally  weak  reactions  are  noted  with  the 
maximum  amount  of  fluid  tested.  Of  course,  in  cases  of 
tabes  and  cerebrospinal  syphilis,  the  smaller  amounts  of 
fluid  show  gradations  in  the  strength  of  the  reaction,  but  a 
diagnosis  of  syphilis  of  the  central  nervous  system  should 
never  be  made  upon  anything  less  than  total  inhibition  of 
hemolysis;  i.e.,  a  double-plus  (four-plus)  reaction,  when 
as  large  an  amount  of  the  serum  as  1  c.c.  is  tested. 

The  writer  has  already  given  the  significance  of  the  posi- 
tive reaction  when  obtained  with  the  different  amounts  of 
the  cerebrospinal  fluid  tested  but  mil  here  summarize  the 
interpretation  to  be  given  the  reaction  in  this  respect : 

If  amounts  of  the  spinal  fluid  as  small  as  0.05  or  0.1  c.c. 
give  total  inhibition  of  hemolysis  the  diagnosis  is  almost 
certainly  paresis;  if  0.2  c.c.  of  the  fluid  give  the  same  re- 
sult, the  diagnosis  is,  in  all  probability,  paresis;  if  larger 
amounts  up  to  0.5  c.c.  of  the  fluid  are  used,  the  diagnosis 
is  probably  paresis ;  while  if  still  larger  amounts  are  used 
the  diagnosis  must  rest  largely  upon  the  clinical  symptoms 
present,  as  cerebrospinal  s}T)hilis,  tabes,  and  paresis  all 
give  positive  results  with  the  larger  quantities  of  fluid. 

In  order  to  illustrate  the  difference  in  results  obtained 
by  using  different  amounts  of  the  cerebrospinal  fluid.  Table 
LIII  is  inserted,  showing  the  results  of  the  Wassermann 
test  upon  cases  of  cerebrospinal  syphilis,  tabes  and  paresis. 

A  consideration  of  this  table  will  show  the  importance 
of  testing  different  amounts  of  the  cerebrospinal  fluid  and 
how  useful  such  titrations  are  in  the  differentiation  of  the 
conditions  mentioned,  especially  the  differentiation  of  pare- 
sis from  cerebrospinal  syphilis  and  tabes. 

It  will  be  understood  that  the  interpretation  of  the  re- 
sults of  the  Wassermann  test  upon  the  cerebrospinal  fluid 


212 


THE   WASSEEMASTF   TEST 


Table  LIII 

The  Eestjlts  of  the  Wassermann  Test  upon  the  Cerebrospinal  Fluid  in 
Cerebrospinal  Syphilis,  Tabes,  and  Paresis 


name  of 

WASSERMANN 
IN  BLOOD 

AMOUNT  OP  CEREBROSPINAL  FLUID  C.C. 

infection 

CASE  NO. 

0.05 

0.1 

0.2 

0.5 

0.7 

1 

Cerebrospinal 
syphilis 
1 
2 
3 
4 
5 
6 
7 
8 

+  + 

+  + 

+ 
+  - 
+  + 

+ 

- 

- 

+ 
+  - 

+  + 

+ 

+  - 

+  - 
+  + 

+ 

+  + 

+  + 

+  + 

+ 

+  + 
+  + 
+  + 
+  + 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 

Tabes 
1 
2 
3 
4 
5 
6 

+  + 
+  + 

+  + 

+ 
+  + 

; 

- 

+ 

+ 
+  + 

+  + 
+ 

+  + 
+  + 

+ 
+  - 

+  + 
+  + 
+  + 
+  + 

+ 

+ 

+  + 
+  + 
+  + 

+  + 

+ 

+  + 

Paresis 
1 
2 
3 
4 
5 
6 
7 
8 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+ 

+  + 

+  + 

+ 

+  + 
+  + 
+  + 
+ 
+  + 

+  + 
+  + 
+  + 
+  + 
+  + 

+  4- 
+  + 
+  + 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 

+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 
+  + 

should  be  based  not  only  upon  this  test  but  also  upon  the 
result  of  the  cell  count,  the  colloidal  gold  reaction  and  the 
tests  for  the  increase  in  globulins,  as  all  should  be  taken 
into  consideration  in  arriving  at  a  just  conclusion  regard- 
ing the  condition  present  in  any  ease,  but  the  Wassermann 
reaction  alone  is  sufficient  to  base  a  diagnosis  of  syphilitic 
infection  of  the  central  nervous  system  upon,  if  positive. 

In  using  the  AVassermann  test  upon  the  cerebrospinal 
fluid  in  the  determination  of  the  cure  of  syphilis  the  re- 
action should  be  negative  with  1  c.c.  of  the  fluid.  If  this 
quantity  of  the  fluid  gives  even  a  partial  positive  result 
the  case  should  not  be  considered  as  cured  but  treatment 
should  be  continued. 

The  early  occurrence  of  involvement  of  the  central  nerv- 


CEREBKOSPIITAL   FLUID  213 

ous  system,  as  shown  by  the  results  of  the  Wassermann 
test,  renders  this  procedure  upon  the  cerebrospinal  fluid  as 
essential  as  upon  the  blood  in  syphilitic  infections,  and  the 
test  should  be  employed  much  more  frequently  than  it  now 
is  as  a  routine  measure.  No  patient  should  be  assured  that 
he  is  free  from  syphilitic  infection  unless  the  cerebrospinal 
fluid  has  been  found  to  give  a  negative  reaction  with  quan- 
tities as  large  as  1  c.c,  but,  on  the  other  hand,  a  diagnosis 
of  involvement  of  the  central  nervous  system  should  not  be 
made  unless  complete  inhibition  of  hemolysis  occurs  with 
this  quantity  of  spinal  fluid. 

The  Wassermann  test  upon  the  blood,  in  cases  showing 
some  nerve  condition,  if  positive,  does  not  prove  that  the 
condition  is  syphilitic  in  nature,  but  if  the  cerebrospinal 
fluid  is  also  positive,  the  condition  is  almost  certainly  syph- 
ilitic. While  the  blood  serum  is  often  negative  in  certain 
syphilitic  conditions  of  the  central  nervous  system,  it  is 
but  rarely  that  the  cerebrospinal  fluid  is  negative  when 
quantities  as  large  as  0.7  to  1  c.c.  are  employed  in  the  test, 
so  that  the  examination  of  the  spinal  fluid  will  detect  many 
syphilitic  infections  that  would  be  overlooked  if  one  de- 
pended entirely  upon  the  result  of  the  Wassermann  test 
upon  the  blood.  For  this  reason  the  writer  urgently  rec- 
ommends the  testing  of  the  cerebrospinal  fluid  in  every  case 
of  syphilis  in  which  it  is  possible  to  do  so  and  this  test 
should  never  be  omitted  in  the  determination  of  the  cure 
of  a  syphilitic  infection.  The  operation  of  lumbar  punc- 
ture, when  properly  performed,  is  not  dangerous  or  very 
painful,  and  the  amount  of  information  that  is  often  se- 
cured by  the  examination  of  the  cerebrospinal  fluid  more 
than  compensates  for  any  discomfort  that  may  be  caused 
by  the  collection  of  the  fluid. 

The  Colloidal  Gold  Test 

The  colloidal  gold  test  was  recommended  by  Lange,"^^^  in 
1913,  as  a  method  of  distinguishing  between  certain  syph- 


214  THE   WASSEEMAFN   TEST 

ilitic  and  nonsyphilitic  diseases  of  the  central  nervous  sys- 
tem, and  today  this  test  stands  as  one  of  the  most  useful 
confirmatory  methods  that  we  possess  in  the  diagnosis  of 
syphilitic  disease  of  this  system. 

The  exact  nature  of  the  reaction  in  the  colloidal  gold  test 
is  unkno^\Ti.  Lange  believes  that  it  is  a  method  which  en- 
ables us  to  measure  the  protein  content  of  the  cerebro- 
spinal fluid,  for  he  found  that  this  fluid  under  conditions 
in  which  the  protein  content  was  increased  caused  the  pre- 
cipitation of  the  gold  solution  and  that,  within  certain 
dilutions,  this  precipitation  was  characteristic  of  syphilitic 
conditions  of  the  central  nervous  system.  Previously,  Zsig- 
mondy^^^  had  demonstrated  that  the  clear  red  color  of 
solutions  of  colloidal  gold,  prepared  first  by  him,  changed 
to  a  blue  with  the  precipitation  of  the  gold  upon  the  ad- 
dition of  electrolytes,  and  that  solutions  of  protein  in  the 
presence  of  an  electrolyte  inhibited  the  precipitate  and  that 
it  was  possible  in  this  way  to  determine  for  various  pro- 
teins the  inhibitive  point  and  thus  differentiate  them. 
Accordingly,  Jaeger  and  Goldstein^-^  regard  the  reaction 
obtained  in  the  colloidal  gold  test  as  a  purely  physical 
reaction  of  an  electrolytic  nature,  but  others,  especially 
Zaloziecki,^^^  believe  it  to  be  an  immunity  reaction.  It  is 
true  that  the  reaction  is  obtained  when  the  protein  content 
of  the  spinal  fluid  is  increased  but  it  is  yet  too  early  to  in- 
sist that  it  is  wholly  due  to  protein  increase  in  the  fluid. 

The  literature  concerning  this  test  is  now  quite  volumi- 
nous and  all  investigators  are  unanimous  in  their  state- 
ments regarding  the  value  of  the  test  in  s^^philis  of  the 
central  nervous  system,  but  there  is  considerable  disagree- 
ment as  to  the  weight  to  be  placed  upon  the  various  findings 
with  the  test  and  their  exact  significance.  However,  all 
agree  that  the  test  is  practically  specific  for  paresis  and 
some  of  those  having  a  large  experience  with  it  insist  that 
a  diagnosis  of  this  condition  may  be  made  from  the  result 
of  this  test  alone.    The  writer  can  not  agree  with  this  opin- 


CEREBROSPINAL   FLUID  215 

ion  of  the  value  of  the  test  in  paresis,  although  it  is  un- 
doubtedly true  that  in  at  least  96  to  98  per  cent  of  cases 
the  typical  paretic  curve  is  obtained,  but  the  same  type  of 
reaction  has  been  observed  in  other  conditions,  although 
very  rarely.  However,  the  fact  that  a  paretic  curve  does 
occur  in  other  conditions  than  paresis  renders  it  impos- 
sible for  the  test  to  be  considered  as  absolutely  specific  of 
this  condition,  although  it  must  be  regarded  as  the  most 
valuable  of  all  confirmatory  evidences  of  the  presence  of 
the  disease. 

Technic  of  the  Test. — The  technic  of  the  colloidal  gold 
reaction  is  very  simple,  but  the  preparation  of  the  solu- 
tion of  colloidal  gold  is  extremely  difficult  and  the  utmost 
care  must  be  used  at  every  stage  of  the  process  or  results 
will  not  be  satisfactory.  In  fact,  it  may  be  said  that  the 
entire  technic  of  the  test  really  consists  in  the  preparation 
of  the  solution  of  colloidal  gold,  for  if  a  good  solution  is  ob- 
tained there  is  little  difficulty  in  performing  the  test. 

Preparation  of  the  Colloidal  Gold  Solution.— It  is  abso- 
lutely requisite  that  all  chemicals  used  in  the  preparation  of 
the  solution  be  chemically  pure  and  the  products  of  Merck 
have  been  found  most  satisfactory  by  numerous  workers. 
The  following  chemicals  are  used  in  the  preparation  of  the 
colloidal  gold: 

1.  Merck's  Gold  Chloride.     Acid. 

2.  Merck's  Blue  Label  Oxalic  Acid.    Crystals. 

3.  Merck's  Blue  Label  Potassium  Carbonate. 

4.  Merck's  Formaldehyde.     Highest  Purity,  40%. 

All  glassware  used  in  making  the  colloidal  gold  solution 
should  be  thoroughly  washed  in  hot  water  with  ivory  soap, 
rinsed  in  tap  water  for  several  minutes;  then  with  hot 
bichromate  solution,  allowing  the  beakers,  pipettes,  and 
test  tubes  to  remain  in  the  bichromate  solution  for  fifteen 
or  twenty  minutes ;  after  which  they  are  rinsed  in  distilled 
water  and  finally  in  triple  distilled  water.  The  glassware 
should  be  used  as  promptly  as  possible  after  cleaning,  the 


216  THE   WASSERMANN   TEST 

pipettes  and  test  tubes  being  dried  in  a  hot  air  oven  before 
use.  Thermometers  used  in  preparing  the  solution  should 
be  cleaned  in  the  same  manner  as  the  glassware. 

The  distilled  water  used  should  be  distilled  in  an  appa- 
ratus in  which  there  are  no  rubber  connections  and  the 
water  should  be  distilled  three  times  before  it  is  used.  In 
certain  regions,  where  the  water  is  very  heavily  impreg- 
nated with  mineral  salts,  it  may  have  to  be  distilled  four, 
or  even  five  times,  and  this  is  also  true  if  the  water  used 
has  been  heavily  impregnated  with  chlorine  in  purifying 
it  for  domestic  use. 

The  following  method  of  preparing  the  colloid  of  gold 
solution  is  recommended  by  Kolmer^-^  who  states  that  it 
is  adapted  from  the  method  of  Miller,  Brush,  Hammers, 
and  Felton.^'° 

1.  Heat  1000  c.c.  of  triply  distilled  water  over  a  proper  burner  in  a  properly 
washed  beaker  containing  a  thermometer. 

2.  When  the  temperature  reaches  60°  C.  add  10  c.c,  of  a  1  per  cent  solution 
of  Merck's  gold  chloride  crystals  in  triply  distilled  water  and  7  c.c.  of  a  2 
per  cent  solution  of  Merck's  blue  label  potassium  carbonate  in  triply  distilled 
water. 

3.  At  80°  C,  while  stirring  briskly,  add  10  drops  of  a  1  per  cent  solution 
of  Merck's  blue  label  oxalic  acid  crystals,  in  triply  distilled  water. 

4.  At  90°  C.  remove  tho  burner  and,  while  stirring,  add  5  c.c.  of  a  solution 
of  1  c.c.  of  Merck's  highest  purity  formaldehyde  in  40  c.c.  of  triply  distilled 
water,  or  enough  to  produce  an  initial  pink  color. 

5.  The  solution  must  be  neutral  in  reaction  when  used  and  to  determine 
the  reaction  it  is  titrated  with  a  1  per  cent  solution  of  alizarin  red,  in  50  per 
cent  alcohol.  With  this  indicator  the  neutral  point  is  shown  by  a  brownish 
red  tint;  an  acid  solution  gives  a  lemon-yellow,  and  an  alkaline  solution  a 
purple-red,  color. 

In  determining  the  reaction  the  following  procedure  is 
carried  out: 

In  a  properly  cleaned  beaker  is  placed  10  c.c.  of  the  colloidal  gold  solu- 
tion and  two  drops  of  the  indicator  are  added.  If  a  brownish  red  tint  appears 
the  solution  is   satisfactory;    if  not,  the   color  is   noted,   and   according   to 

N  N 

whether  it  is  acid  or  alkaline  it  is  titrated  with  either    -zrx   NaOH  or    -^^    HCl. 

50  50 

If  acid  the  NaOH  is  used  and  if  alkaline  the  HCl.  When  the  neutral  point 
is  reached;  i.e.,  a  brownish  red  tint  appears,  the  amount  of  reagent  n^ces- 


CEREBROSPINAL   ELUID  217 

sary  to  neutralize  the  10  c.c.  of  the  gold  solution  is  noted  and  the  amount 
necessary  to  neutralize  1000  c.c.  is  readily  calculated.  Neutralization  should 
be  made  with  normal  or  decinormal  solutions  of  the  acid  or  alkali,  as  the 
case  may  be. 

Kolmer  gives  the  following  standards  for  an  efficient  col- 
loidal gold  solution: 

1.  It  must  be  absolutely  transparent  and  of  a  brilliant  red  orange  or  sal- 
mon red  color.  (The  writer  has  found  that  if  there  is  the  least  trace  of 
cloudiness  in  the  solution,  it  will  be  found  unserviceable.) 

2.  Five  cubic  centimeters  of  the  solution  should  be  completely  precipitated 
in  one  hour  by  1.7  c.c.  of  a  1  per  cent  solution  of  sodium  chloride  in  distilled 
water. 

3.  The  solution  must  be  neutral  in  reaction  when  used. 

4.  The  solution  must  not  produce  a  reaction  greater  than  a  red  blue  dis- 
coloration (No.  1  reaction)  with  normal  cerebrospinal  fluid,  and  should  give 
a  typical  curve  with  a  known  paretic  fluid. 

To  the  above  the  writer  would  add  that  the  colloidal  gold 
solution  should  show  no  evidence  of  having  been  deposited 
on  the  glass  flask  in  which  it  is  stored  or  any  evidence  of 
a  precipitate  within  the  flask.  It  has  been  noted  by  the 
writer  that  some  glassware  is  entirely  unsuitable  for  use 
with  colloidal  gold  as  it  apparently  is  attacked  by  it  with 
a  resulting  precipitate  in  the  solution  or  deposited  upon 
the  sides  and  bottom  of  the  storage  flask.  Jena  glassware 
is  the  best  but  Pyrex  has  been  found  satisfactory.  The 
colloidal  gold  should  be  kept  in  the  flask  in  the  dark  and 
will  keep  for  months  unchanged  if  properly  handled. 

The  fact  that  the  colloidal  gold  solution  is  perfectly  clear 
does  not  indicate  that  it  will  prove  effective  in  actual  prac- 
tice for  it  has  been  found  that  perfectly  clear  solutions  are 
sometimes  worthless.  Such  solutions  are  called  "pro- 
tected solutions"  because  no  precipitation  will  take  place 
after  the  addition  of  any  electrolyte,  while  "unprotected 
solutions,"  as  they  are  called,  act  efficiently  in  the  test, 
and  are  those  that  can  be  completely  precipitated  by  1.7 
c.c.  of  a  1  per  cent  sodium  chloride  solution  in  one  hour. 
This  test  of  the  efficiency  of  a  colloidal  gold  solution  is  very 
important  and  should  always  be  carried  out  before  any 


218  THE   WASSERMANN   TEST 

such  solution  is  depended  upon  for  use  in  the  colloidal 
gold  test.  It  has  also  been  determined  that  alkaline  col- 
loidal gold  solutions  are  practically  inert  when  used  with 
a  positive  cerebrospinal  fluid,  while  acid  solutions  give 
little  reaction  with  a  positive  fluid,  and  an  abnormal  reac- 
tion with  a  normal  spinal  fluid.  If  the  colloidal  gold  so- 
lution is  only  slightly  acid  it  will  react  well  with  paretic 
fluids,  but  will  give  a  reaction  with  normal  fluids  similar  to 
that  obtained  in  the  luetic  zone  with  fluid  from  some  syph- 
ilitic conditions.  In  order  to  secure  reliable  results  the 
colloidal  gold  solution  must  he  neutral  in  reaction. 

Method  of  Performing  the  Test. — The  technic  of  the  col- 
loidal gold  test  is  very  simple  but  to  secure  proper  results 
each  step  must  be  carefully  performed.  All  glassware 
must  be  prepared  as  already  described  and  pipettes  must 
be  perfectly  dry  before  being  used. 

Eleven  test  tubes,  cleaned  as  described  and  perfectly  dry, 
are  arranged  in  a  rack  and  in  the  first  tube  is  placed  1.8 
c.c.  of  freshly  prepared,  sterile  0.4  per  cent  sodium  chlo- 
ride solution  prepared  from  chemically  pure  sodium  chlo- 
ride. Into  each  of  the  ten  remaining  test  tubes  place 
1  c.c.  of  the  salt  solution.  With  a  dry  pipette  add  0.2  c.c. 
of  the  cerebrospinal  fluid  to  be  tested,  which  must  be  free 
from  blood,  to  the  first  tube  and  mix  thoroughly.  After 
mixing,  1  c.c.  of  the  mixture  is  transferred  to  the  second 
tube,  mixed,  and  1  c.c.  of  this  mixture  transferred  to  the 
third  tube,  and  this  process  repeated  until  the  tenth  tube 
is  filled,  when  from  that  tube  1  c.c.  is  discarded.  The  elev- 
enth tube  acts  as  a  control  and  will  contain  no  cerebro- 
spinal fluid.  The  dilutions  which  have  thus  been  made  are 
as  follows :  1-10,  1-20, 1-40,  1-80,  1-160,  1-320,  1-640, 1-1280, 
1-2560,  and  1-5120. 

After  the  dilutions  of  the  cerebrospinal  fluid  have  been 
made,  as  indicated,  there  is  added  to  each  tube  5  c.c.  of  the 
colloidal  gold  solution.  This  is  thoroughly  mixed  with  the 
fluid  in  the  tubes  and  the  rack  kept  at  room  temperature 


l:IO         J:20       I'-HO       IsSO       |;l60     1:320     \-6H0    |:  1280   1  =  2560  U5I20   Control 


5       ^ 


i 


Parefic      Reaction. 

I-IO         |i20        1-^0        |:80        J-I60      1^320      1:640      t:l280    |:2560  |:5120  Control 


^^^s^   V^iix 


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V""""^-^y 


I  g-  --^s? 


Tabetic    Reaction. 


Plate  III. 

Paretic  Reaction. — The  upper  row  of  tubes  illustrates  the  reaction,  or  curve,  obtained 
with  the  colloidal  gold  test  in  the  cerebrospinal  fluid  of  paresis  and  is  known  as  the 
paretic  curve  or  reaction. 

Tabetic  Reaction. — The  lower  row  of  tubes  illustrates  the  reaction,  or  curve,  obtained 
with  the  colloidal  gold  test  in  the  cerebrospinal  fluid  of  tabes  and  is  known  as  the  tabetic 
reaction.     (L,uetic-zone  curve.) 


CEREBROSPINAL   FLUID  219 

overnight,  the  test  being  read  upon  the  next  day  and  re- 
.  corded  by  the  numbers  corresponding  to  the  changes  in  the 
color  of  the  mixture  observed,  as  follows : 

5.  Complete  precipitation.  The  supernatant  liquid  is 
colorless. 

4.  Pale  blue. 

3.  Blue. 

2.  Lilac  or  purple. 

1.  Eed-blue. 

0.  No  change. 

Reading  the  Results. — The  results  of  the  test  are  read  by 
the  numbers  corresponding  to  the  color  changes,  as  noted 
in  the  preceding  table.  Thus,  if  the  first  four  tubes  show 
complete  precipitation,  with  a  colorless  supernatant  fluid, 
the  fifth  and  sixth  tubes  a  pale  blue  color,  the  seventh  tube 
lilac  or  purple,  and  the  remaining  tubes  no  change,  the 
reaction  would  be  read  as  follows :  5555442000. 

The  Results  of  the  Test  in  Vaxious  Syphilitic  Condi- 
tions.— The  change  in  color  in  the  various  tubes  will  differ 
with  different  syphilitic  conditions  of  the  central  nervous 
system,  the  color  reaction  with  paresis  being  the  most  char- 
acteristic and  diagnostic  of  all,  and  the  changes  observed 
are  called  ''curves."  Thus,  we  have  a  "paretic  curve"  and 
a  "luetic  zone  curve,"  as  well  as  a  "meningitic  zone  curve." 

The  "Paretic  Curve." — The  reaction  observed  in  the 
cerebrospinal  fluid  in  paresis  is  so  constant  and  typical 
that  it  may  be  considered  as  the  most  valuable  of  all  the 
confirmatory  signs  that  we  possess  of  this  type  of  involve- 
ment of  the  central  nervous  system.  It  is  to  this  type  of 
the  reaction  that  Miller  and  Levy^^^  gave  the  name  "pa- 
retic curve ' '  and  it  is  this  type  of  reaction  that  is  met  with 
in  practically  100  per  cent  of  cases  of  paresis.  In  report- 
ing the  colloidal  gold  test  it  is  usually  customary  to  record 
the  reaction  upon  a  chart  and  when  this  is  done,  the  pa- 
retic curve  will  appear  substantially  as  in  Table  LIV. 


220 


THE   WASSERMANF   TEST 


Table  LIV 


Illustrating  Type  of  Reaction  Obtained  with  the  Colloidal  Gold  Test 
IN  Paresis  (Paretic  Curve) 

COLOR            1  color] dilutions  OF  CEREBROSPINAL  FLUID  AVITH  0.4  PER  CENT  NACL 

reactions     1    NO.   |1-10|1-20|1-40|1-80|1-160|1-320|1-640|1-1280|1-256D|1-5120 

Colorless           |     5     |    •— |-. -|- ,-|-»-|  —  r -f-»^  |           |             |             | 

Pale  blue          |     4     j        |        |        |        |          |          Pv    1            1            1 

Blue                   |3|||||           1           l^fyl             1 

JLiilac  or  purple  |2|         |         |         |         |           |           |           l*s^l             1 

Eed-blue           |     1     |        |        (        |        1          (          |          |            1^ -^    1 

No  change        |0||||t          1           1           1             1            !*• 

Kecorded  in  numbers  this  reaction  would  be  thus  re- 
ported: 5555554210.  Often  the  first  three,  four,  or  five 
tubes  are  decolorized,  while  the  remaining  ones  show  some 
changes,  as  would  be  found  in  the  following  combinations : 
5554432200;  5555422100,  5555542210,  or  as  many  as  eight 
of  the  tubes  may  be  completely  decolorized.  Curves  of  this 
type  are  characteristic  of  paresis  and  are  not  found  in 
other  SAT3hilitic  diseases  of  the  central  nervous  system, 
although  a  few  reports  are  on  record  of  cases  of  menin- 
gitis and  multiple  sclerosis  giving  similar  curves  with  this 
test.    A  paretic  curve  is  also  observed  in  taboparesis. 

The  Luetic-Zone  Curve. — The  luetic-zone  curve  is  ob- 
tained in  cases  of  cerebrospinal  syphilis  and  tabes,  as  well 
as  in  the  fluid  in  the  tertiary  stage  of  syphilis.  In  tabes 
the  colloidal  gold  test  usually  gives  a  luetic-zone  curve  of 
No.  4  intensity,  the  precipitation  being  only  partial  in  the 
first  two  or  three  tubes,  complete  in  the  next  two  or  more 
tubes,  and  gradually  shading  off  into  normal.  However,  it 
should  be  distinctly  understood  that  there  is  nothing  char- 
acteristic in  the  curves  noted  in  tabetic  patients  and  there 
is  no  constancy  in  the  type  of  curve  observed,  except  that 
the  "paretic  curve"  is  never  noted.  Table  LV  illustrates 
the  "luetic-zone  curve"  obtained  in  a  case  of  tabes. 

Kecorded  by  numbers  the  above  reaction  would  be  thus 
reported:  4445554200.  In  cerebrospinal  syphilis  the  fluid 
usually  shows  weaker  reactions  than  that  for  tabes  but  al- 


CEREBROSPINAL   FLUID 


221 


Table  LV 

Illustrating  Type  of  Reaction  Obtained  witij  the  C()lloidal  Gold  Test 
IN  Tabes  (Luetic  Zone  Curve) 


color         I  color]  dilutions  op  cerebrospinal  fluid  with  0.4  PER  cent  nacl 

REACTIONS     1    NO.   1 1-10|  1-20|  1-40|  1-80|  1-160|1-320|1-640|1-1280|1-2560|1-5120 

Colorless            I     5     1         1         1         U  •"!"•  •""!" 'v  1           1             1             1 

Paleblue          j     4     |    •^_,.|«,''|         |           |           f^*^  I             |             | 

Blue                  1     3     1         1         1         1         1           1           1         1,          1     •       1 

Lilac  or  purple  1     2     |         |         |         |         |           |           |           1   ^•v     1             1 

Red-blue           1     1     1        1        t                 1          1          1          1         "^L           1 

No  change         1     0     I         1         1         1         I           1           I           I             |  '^  • — f— • 

ways  in  the  luetic-zone,  while  the  reactions  obtained  in 
the  fluid  during  the  tertiary  stage  of  syphilis  when  symp- 
toms of  involvement  of  the  central  nervous  system  are 
absent  are  still  weaker,  only  slight  changes  being  observed, 
as  a  rule,  in  the  luetic  zone  of  the  test. 

In  nonsyphilitic  disease  of  the  central  nervous  system, 
as  in  meningitis,  acute  poliomyelitis,  multiple  sclerosis, 
etc.,  the  findings  with  the  colloidal  gold  test  are  often  con- 
fusing and  of  little  practical  value.  In  meningitis  the  so- 
called  '^meningitic  zone"  curve  is  encountered,  in  which 
the  color  changes  occur  in  the  highest  dilutions,  complete 
precipitation  occurring  at  the  opposite  end  of  the  chart 
from  paresis.  Table  LVI  illustrates  the  type  of  curve  ob- 
tained in  tuberculous  meningitis  or  purulent  meningitis. 

Table  LVI 

Illustrating  Type  of  Reaction  Obtained  with  the  Colloidal  Gold  Test 
IN  Meningitis  (Meningitic  Curve) 


COLOR             1  color] DILUTIONS  OP  CEREBROSPINAL  FLUID  WITH  0.4  PER  CENT  NACL 

reactions     I    NO.   |1-10|1-20|1-40|1-80|1-160|1-320  1-640|1-1280|1-2560|1-5120 

Colorless            I     5     |         |         |         |         |           j                      |     ,— f".      | 

Paleblue           |     4     |         |         |         |         |           |                    J^          1        x    1 

Blue                  1     3     1         1        1         1         1           1             ,.'  1             1          \| 

Lilac  or  purple  1     2     |         |         |         |         |^._|_.^'           |             |             fy^ 

Red-blue           1     1     1         1         1         L.I           1           1           1             1             |N 

No  change         (     0     |   ._|-.^|_,'|         1           |           |           I             I             1      • 

Recorded  by  numbers  the  above  reaction  would  be  thus 
reported:  0001223550. 


222  THE   WASSERMAlSriSr   TEST 

Interpretation  of  the  Results  of  the  Colloidal  Gold 
Test. — There  is  only  one  condition  in  whicli  the  colloidal 
gold  test  may  be  practically  relied  upon  for  diagnosis  and 
that  is  paresis.  In  the  overwhelming  number  of  instances, 
if  a  typical  paretic  curve  is  obtained  with  this  test  it  is 
safe  to  make  a  diagnosis  of  this  condition  and  the  Wasser- 
mann  test  upon  the  blood  and  cerebrospinal  fluid  will  gen- 
erally confirm  the  diagnosis.  However,  the  writer  is  still 
doubtful  if  we  should  rely  upon  the  colloidal  gold  reaction 
alone  in  making  a  diagnosis  of  paresis,  especially  if  the 
other  diagnostic  aids  that  we  possess  do  not  confirm  the 
reaction.  It  must  be  admitted,  however,  that  the  first  evi- 
dence we  have  in  many  cases  of  incipient  paresis  is  a  pos- 
itive result  mth  this  test  and  whenever  such  a  result  is 
obtained  it  should  be  given  great  weight  and  the  most  in- 
tensive specific  treatment  at  once  instituted. 

In  cerebrospinal  syphilis  and  tabes  the  colloidal  gold  re- 
action is  of  no  diagnostic  importance  and  the  results 
obtained  should  never  be  considered  as  more  than  confir- 
matory of  the  diagnosis  in  cases  which  are  very  suspicious 
in  other  respects.  With  the  exception  of  paresis  and  tabo- 
paresis the  colloidal  gold  test  is  of  value  only  as  an  aid  in 
the  diagnosis  of  syphilis  of  the  central  nervous  system.  In 
congenital  syphilis,  cerebrospinal  syphilis,  tabes,  and  sec- 
ondary and  tertiary  syphilis  the  results  are  inconstant 
and  only  of  confirmatory  value,  but  its  appearance  in  pare- 
sis, in  mau}^  cases,  before  symptoms  of  the  condition  de- 
velop, renders  the  test  of  great  value  in  early  diagnosis 
and  as  this  complication  may  occur  in  any  case  of  syph- 
ilitic infection  the  routine  testing  of  the  cerebrospinal  fluid 
with  the  colloidal  gold  test  is  to  be  encouraged. 

The  Cell  Count 

It  is  well  knoANTi  that  in  most  inflammatory  conditions 
of  the  central  nervous  system  there  is  an  increase  in  the 
number  of  cells  found  in  the  cerebrospinal  fluid  and  this 


CEREBROSPIlSrAL   FLUID  223 

increase  occurs  to  such  an  extent  in  some  conditions  as  to 
be  of  considerable  service  in  their  differential  diagnosis. 
The  cell  count,  however,  so  far  as  syphilitic  disease  of  the 
central  nervous  system  is  concerned,  is  seldom  of  more 
than  confirmatory  value  of  results  obtained  by  other  meth- 
ods of  laboratory  diagnosis.  In  no  syphilitic  condition  of 
the  central  nervous  system  is  the  cell  count  specific  nor 
can  one  differentiate  syphilitic  disease  from  other  diseases 
of  this  system  by  its  means. 

An  increase  in  the  number  of  cells  in  the  cerebrospinal 
fluid,  called  pleocytosis,  occurs  in  practically  95  per  cent 
of  syphilitic  affections  of  the  central  nervous  system  but 
also  occurs  in  other  diseases  of  this  system,  and  in  acute 
infections,  as  cerebrospinal  meningitis,  although  in  non- 
syphilitic  conditions  the  cell  count  is  seldom  as  high  as  in 
conditions  like  tabes,  cerebrospinal  syphilis,  paresis,  and 
syphilitic  meningitis.  According  to  Nonne^^^  the  cell  count 
in  the  cerebrospinal  fluid  may  be  appreciably  increased  in 
from  30  to  40  per  cent  of  cases  of  syphilis  even  in  the  ab- 
sence of  symptoms  of  organic  nervous  disease.  In  old 
cases  of  tabes,  or  cases  in  which  the  symptoms  are  not  pro- 
gressive, there  may  be  an  absence  of  pleocytosis  and  even 
in  progressive  cases  this  may  sometimes  occur. 

Efforts  have  been  made  to  differentiate  the  kinds  of  cells 
increased  in  the  pleocytosis  of  syphilitic  disease  of  the  cen- 
tral nervous  system,  and  Alzheimer  has  rendered  it  pos- 
sible to  recognize  the  different  types  of  cells  that  occur. 
However,  from  a  practical  standpoint,  the  differentiation 
of  the  various  cells  found  is  of  very  little,  if  any,  assistance 
in  differentiating  the  various  syphilitic  diseases  of  the 
nervous  system,  a  simple  count  of  the  cells  present  being 
as  useful  as  any  differential  count.  As  Nonne  well  says  :^^^ 
''The  attempt  to  recognize  in  the  spinal  fluid  a  character- 
istic cell-picture  for  the  development  of  a  paresis,  or  to 
foretell  the  time  of  progression  of  a  syphilogenetic  affec- 
tion, has  not  as  yet  been  crowned  with  success." 


224  THE   WASSERMANN   TEST 

The  cells  which  are  increased  in  syphilitic  disease  of  the 
central  nervous  system  comprise  both  lymphocytes  and 
leucocytes,  but  their  origin  is  not  absolutely  determined. 
Whether  they  originate  from  the  blood  or  from  the  connec- 
tive tissue  of  the  leptomeninges  is  unsettled  but  the  pleo- 
cytosis  is  the  direct  result,  undoubtedly,  of  an  inflamma- 
tory process  in  the  leptomeninges.  This  is  the  conclusion 
reached  by  ISTonne  and  it  is  believed  that  it  is  the  one  gen- 
erally held  by  the  best  students  of  the  subject. 

Technic  of  Counting  the  Cells. — In  counting  the  cells  in 
the  cerebrospinal  fluid  the  most  generally  used  method  is 
with  the  Fuchs-Eosenthal  counting  chamber.  The  method 
of  making  the  dilution  of  spinal  fluid  and  counting  is  as 
follows:  The  staining  fluid  consists  of  0.1  gram  gentian 
violet;  2.0  grams  glacial  acetic  acid;  and  50  c.c.  distilled 
water.  This  fluid  is  drawn  up  into  the  ordinary  pipette 
used  for  counting  the  white  cells  of  the  blood,  to  the  point 
marked  I  and  then  the  cerebrospinal  fluid  drawn  up  to  the 
point  marked  II.  The  pipette  is  now  shaken  very  thor- 
oughly for  at  least  two  minutes,  when  a  drop  of  the  mix- 
ture is  placed  on  the  counting  chamber,  a  few  drops  being 
expelled  from  the  pipette  before  doing  so.  All  of  the 
lymphocytes  and  leucocytes  are  counted  in  all  the  squares 
and  the  total  number  divided  by  three.  Nonne  regards 
from  0  to  5  cells  as  normal ;  6  to  10  as  borderland  cases ; 
and  more  than  10  cells  as  pathological. 

The  Fuchs-Eosenthal  counting  chamber  is  not  necessary 
in  making  a  count  of  the  cells  in  the  spinal  fluid,  as  the 
Zappert,  Turk,  or  Le\^  counting  chamber  may  be  used 
just  as  well,  the  spinal  fluid  being  undiluted  or,  if  the  cells 
are  very  numerous,  diluted  as  for  making  a  white  blood 
cell  count. 

The  staining  of  the  cells  is  not  necessary  if  the  fluid  be 
diluted  with  a  0.5  per  cent  solution  in  distilled  water  of 
glacial  acetic  acid,  when  the  Fuchs-Rosenthal  chamber  for 
counting  is  used,  while  if  either  the  Turk  or  Zappert  count- 


CEREBROSPIISrAL   FLUID 


225 


ing  chambers  are  used  the  fluid  should  be  used  undiluted, 
unless  the  fluid  is  purulent  or  contains  an  excessive  num- 
ber of  cells. 

The  number  of  cells  found  in  the  cerebrospinal  fluid  in 
the  various  syphilitic  diseases  of  the  nervous  system  varies 
very  greatly  and  no  diagnostic  data  can  be  based  upon  the 
number  of  the  cells  alone.  The  increase  in  the  number  of 
cells  is  the  important  diagnostic  feature,  but  this  increase 
is  not  of  such  a  nature  that  one  can  say  that  paresis  exists 
in  this  case,  and  tabes  in  that.  In  illustration  of  this. 
Table  LVII  shows  the  counts  observed  in  paresis,  cerebro- 
spinal syphilis,  and  tabes. 

Table  LVII 

Cells  Counts  in  the  Cerebrospinal  Fluid  Observed  in  Paresis,  Cerebro- 
spinal Syphilis,  and  Tabes 


paresis 

cerebrospinal  syphilis 

TABES 

case  no. 

COUNT 

CASE  NO. 

COUNT 

CASE  NO. 

COUNT 

1 

18 

1 

13 

1 

9 

2 

45 

2 

64 

2 

8 

3 

67 

3 

243 

3 

43 

4 

16 

4 

11 

4 

44 

5 

29 

5 

86 

5 

55 

6 

88 

6 

65 

6 

69 

7 

12 

7 

50 

7 

186 

8 

36 

8 

590 

8 

237 

9 

168 

9 

344 

9 

36 

10 

243 

10 

156 

10 

110 

11 

507 

11 

6 

11 

124 

12 

323 

12 

402 

12 

116 

It  will  be  seen  from  the  above  table  that  there  is  nothing 
characteristic  in  the  number  of  cells  found  in  the  cerebro- 
spinal fluid  in  the  three  conditions,  the  characteristic  fea- 
ture being  that  there  is  an  increase  above  normal  in  all  the 
cases  cited. 

Interpretation  of  Results  of  Count. — The  increase  in  the 
number  of  cells  in  the  cerebrospinal  fluid,  as  has  been 
stated,  is  due  to  inflammatory  conditions  of  the  leptomen- 
inges,  but  an  increase  does  not  always  indicate  that  the 
condition  causing  it  is  syphilitic.     Of  the  syphilitic  con- 


226  THE   WASSERMANN   TEST 

ditions  of  the  central  nervous  system  the  highest  counts 
are  generally  observed  in  syphilitic  meningitis,  the  count 
frequently  exceeding  one  thousand  cells;  in  tabes  the 
counts  are  apt  to  be  high,  running  into  the  hundreds;  in 
paresis,  the  average  count  is  not  above  100,  generally  run- 
ning between  20  and  60,  but  counts  above  100  are  often  ob- 
tained, and  the  same  statement  is  true  of  cases  of  cerebro- 
spinal syphilis.  However,  the  mere  number  of  cells  found 
is  no  criterion  of  the  condition  present,  and  the  cell  count 
has  to  be  interpreted  in  conjunction  with  the  clinical  symp- 
toms present  and  the  results  of  the  other  methods  of  lab- 
oratory diagnosis  in  syphilis ;  i.  e.,  the  Wassermann  test, 
the  colloidal  gold  test,  and  the  test  for  increase  in  proteins 
in  the  cerebrospinal  fluid. 

Tests  for  Increase  in  Globulins  in  the  Cerebrospinal 
Fluid. — In  syphilitic  disease  of  the  central  nervous  system, 
as  well  as  in  other  organic  diseases  of  this  system,  the  pro- 
tein content  of  the  cerebrospinal  fluid  is  generally  increased, 
and  this  increase  can  be  detected  by  tests  which  have  been 
perfected  for  demonstrating  an  increased  globulin  content 
in  this  fluid.  Three  methods  are  in  general  use,  kno^vn  as 
the  Nonne  and  Apelt  method,  the  Noguchi  method,  and  the 
Pandy  method. 

The  Nonne-Apelt  Globulin  Test. — This  test  is  thus  de- 
scribed by  Nonne :^^*  "The  simplest  method  of  testing  the 
cerebrospinal  fluid  for  albuminous  bodies  is  to  search  for 
an  increase  of  globulin — the  so-called  ^ Phase  I'  reaction, 
introduced  by  Apelt  and  myself.  For  this  purpose  there  is 
added  to  a  hot  saturated  solution  of  sulphate  of  ammo- 
nium, which  has  been  permitted  to  cool,  an  equal  amount 
of  cerebrospinal  fluid;  1  c.c.  of  each  is  quite  sufficient.  It 
is  advisable  to  pour  one  liquid  gently  on  top  of  the  other; 
if  the  globulins  are  increased,  there  occurs  a  more  or  less 
distinct  gray  ring  at  the  plane  of  contact.  After  this  pre- 
liminary observation  the  mixture  is  well  shaken  and  the 
result  may  be  read  off  within  three  minutes.    If  it  is  dis- 


CEREBEOSPIN'AL   FLUID  227 

tinctly  opalescent  or  cloudy,  we  call  it  a  positive  'Phase  I' 
reaction. ' ' 

In  making  the  above  test  it  is  best  to  add  the  cerebro- 
spinal fluid  from  a  1  c.c.  pipette  very  slowly  to  the  ammo- 
nium sulphate  solution,  as  in  this  way  the  gray  ring  indi- 
cating increased  globulin  is  rendered  much  more  distinct 
than  when  one  fluid  is  poured  into  the  other.  This  method 
is  still  in  general  use  but  has  been  very  largely  displaced 
by  the  Noguchi  and  Pandy  methods. 

The  Noguchi  Butyric  Acid  Test  for  Globulin. — This 
method  was  devised  by  Noguchi,^^^  is  simple,  and  accu- 
rate, and  the  one  that  the  writer  has  generally  used  in  his 
laboratories.  The  method  of  making  the  test  is  thus  de- 
scribed by  Noguchi  '/^^ 

"Two  parts  of  the  cerebrospinal  fluid  to  be  examined 
are  mixed  with  5  parts  of  a  10  per  cent  butyric  acid  solu- 
tion in  physiological  salt  solution,  and  are  heated  over  a 
flame  and  boiled  for  a  brief  period.  One  part  of  a  normal 
solution  of  NaOH  is  then  added  quickly  to  the  heated  mix- 
ture, and  the  whole  boiled  once  more  for  a  few  seconds. 
The  actual  quantities  of  these  three  reagents  that  I  pre- 
fer are  0.1  or  0.2  c.c.  of  the  spinal  fluid,  0.5  c.c.  of  the 
butyric  acid  solution,  and  0.1  c.c.  of  the  normal  sodium 
hydrate.  It  is  necessary  to  take  the  precaution  to  employ 
for  this  test  only  cerebrospinal  fluid  entirely  free  from 
blood." 

A  positive  reaction  with  this  test  is  indicated  by  the  ap- 
pearance of  a  granular  or  flocculent  white  or  gray  precip- 
itate which  settles  slowly  to  the  bottom  of  the  test  tube. 
The  amount  of  this  precipitate  and  the  rapidity  with  which 
it  is  formed  will  vary,  as  Noguchi  observed,  with  the  quan- 
tity of  protein  in  the  fluid  examined,  the  greater  the 
amount  of  protein  the  quicker  the  reaction  will  appear  and 
the  greater  the  amount  of  precipitate,  in  the  one  case  the 
reaction  appearing  within  a  few  minutes,  while,  if  the 
amount  of  protein  increase  is  small  the  reaction  may  take 


228  THE   WASSEEMANN   TEST 

an  hour  or  more  to  appear.  Two  hours  should  be  the  time 
limit  for  the  reaction. 

The  Pandy  Method  for  Globulin  Increase. — A  method 
which  is  spoken  very  highly  of  by  those  who  have  used  it 
is  the  Pandy  method.  It  is  the  most  simple  of  all  methods 
that  have  so  far  been  devised  for  the  purpose  and  is  un- 
doubtedly reliable.  Miller/"  who  has  had  a  large  experi- 
ence with  this  method  thus  describes  its  use: 

' '  Pandy 's  test  has  not  received  the  attention  it  deserves. 
None  of  the  other  reactions  used  to  reveal  an  excess  of 
globulin  is  so  simple  in  execution  or  so  quickly  decisive  in 
its  results.  The  reagent  consists  of  a  saturated  aqueous 
solution  of  carbolic  acid;  ten  parts  of  pure  crystals  are 
added  to  100  parts  of  hot  distilled  water;  the  mixture  is 
kept  at  room  temperature  for  three  or  four  days,  during 
which  time  it  should  be  frequently  shaken.  At  the  end  of 
this  time  the  clear  supernatant  fluid  is  dra^\T.i  off  into  an- 
other bottle.  To  approximately  1  c.c.  of  the  reagent  is 
added  one  drop  of  the  spinal  fluid.  Normally  no  change 
occurs  or  at  most  an  extremely  faint  opalescence;  mth  a 
fluid  abnormal  in  its  protein  content  there  develops  in- 
stantly at  the  point  of  contact  a  bluish  white  cloud,  often 
resembling  a  ring  of  smoke,  which  gradually  settles  to  the 
bottom  of  the  tube." 

The  writer  has  had  very  little  experience  with  this  test 
but  it  has  proved  accurate  in  those  cases  in  which  it  has 
been  used. 

Interpretation  of  the  Results  of  the  Globulin  Tests. — In 
organic  disease  of  the  central  nervous  system  an  increase 
in  the  protein  content  of  the  cerebrospinal  fluid  commonly 
occurs  and  this  increase  is  present  in  the  vast  majority  of 
cases  of  syphilitic  disease  of  this  system.  However,  as 
this  protein  increase,  indicated  by  the  increase  in  globu- 
lins, is  not  confined  to  syphilitic  disease,  a  positive  reac- 
tion with  any  of  the  tests  mentioned  does  not  prove  that 
the  condition  present  is  syphilitic  nor  is  it  possible  to  dif- 


CEREBKOSPIl^AL   FLUID 


229 


ferentiate  the  various  syphilitic  diseases  of  the  central 
nervous  system  by  their  use.  The  tests  are  often  positive 
in  infections  of  the  meninges  due  to  the  pneumococcus,  tu- 
bercle bacillus,  influenza  bacillus,  meningococcus,  and  in 
acute  poliomyelitis,  as  well  as  in  syphilitic  disease,  and 
are  also  present  in  chronic  organic  disease  of  the  central 
nervous  system,  so  that  a  positive  reaction  can  only  be  re- 
garded as  confirmatory  of  the  results  of  other  laboratory 
methods  of  diagnosis  of  syphilitic  involvement  of  this  sys- 
tem and  of  the  nature  of  the  clinical  symptoms  which  may 
be  present. 

Summary  of  the  "Four  Tests." — In  diagnosing  and  in 
differentiating  syphilitic  disease  of  the  central  nervous 
system,  it  is  necessary  to  carefully  consider  the  results  of 
the  ''Four  tests"  as  they  may  be  called,  upon  the  cerebro- 
spinal fluid.  These  are  1,  the  Wassermann  test  upon  the 
fluid ;  2,  the  colloidal  gold  test ;  3,  the  cell  count ;  4,  the  test 
for  increase  in  globulins.  As  an  illustration  of  the  results 
that  are  commonly  obtained  with  these  four  tests,  Table 
LVIII,  adapted  from  Miller^^^  is  inserted: 

Table  LVIII 

Showing  Average  Frequency  of  the  Various  Eeactions  in  Syphilis  of  the 
Central  Nervous  System 


TEST 

PARESIS 
% 

TABES 
% 

CEREBROSPINAL 
SYPHILIS 

% 

Spinal  tiuid  Wassermann 
Globulin  test 
Cell  count 
Colloidal  gold  test 

97 

100 

98 

98-100 

60-89 
90-95 
85-90 
85-90 

85-90 
90-95 
85-90 
85-90 

Paretic 
curve 

Luetic 
curve 

Luetic 
curve 

The  blood  Wassermann  in  these  diseases  gives  approx- 
imately the  following  results:  Paresis,  98-100  per  cent; 
tabes,  70  per  cent;  and  cerebrospinal  syphilis,  70-80  per 
cent.  A  careful  study  of  the  cerebrospinal  fluid  by  means 
of  these  four  reactions  will  often  result  in  enabling  one  to 
differentiate  the  various  syphilitic  diseases  of  the  central 


230  THE   WASSEKMANN"   TEST 

nervous  system  and,  in  tlie  vast  majority  of  onr  cases,  if 
the  tests  are  negative,  to  exclude  syphilitic  disease  in  a 
suspected  individual.  If  the  globulin  test  is  negative  in  a 
patient  suffering  from  disease  of  the  central  nervous  sys- 
tem it  is  almost  certain  that  the  condition  is  not  syphilitic, 
although,  if  positive,  it  can  not  be  said  that  it  is  syphilitic. 
A  fluid  showing  a  positive  Wassermann  reaction  in 
amounts  as  small  as  0.2  c.c,  together  with  a  paretic  curve 
with  the  colloidal  gold  test,  is  sufficient  upon  which  to 
base  a  diagnosis  of  paresis,  while  a  high  cell  count,  a 
luetic  curve  with  the  colloidal  gold  test,  and  a  negative 
Wassermann,  or  one  only  positive  in  large  amounts  of  the 
spinal  fluid,  would  point  to  tabes.  By  a  careful  compar- 
ison of  the  results  of  these  various  tests  w^ith  the  clinical 
symptoms  present  it  should  be  possible,  in  the  vast  ma- 
jority of  instances,  to  make  a  diagnosis  of  the  form  of 
syphilitic  disease  of  the  central  nervous  system  which  may 
be  present,  and  the  value  of  these  tests  in  diagnosing  very 
early  involvement  of  this  system  has  already  been  empha- 
sized. 

In  no  case  can  it  be  stated  that  the  syphilitic  patient  has 
been  given  the  best  that  it  is  possible  for  medical  science 
to  give  him,  either  in  the  way  of  diagnosis  or  treatment, 
if  the  thorough  examination  of  the  cerebrospinal  fluid  has 
been  omitted  and  this  examination  should  become  as  much 
of  a  routine  in  the  diagnosis  and  treatment  of  syphilis  as 
is  the  AYassermann  test  upon  the  blood  serum. 


BIBLIOGRAPHY 


The  references  given  below  do  not  pretend  to  cover  the  literature  of  the 
subject,  which  is  now  immense,  but  only  cover  the  papers  actually  referred  to 
in  the  text. 

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2Fodor,  von:     Deutsch.  med.  Wchnschr.,  1888,  vi,  460. 

sBuchner:     Centralbl.  f.  Bakt.,  1889,  v,  252. 

4Buchner:     Arch.  f.  Hyg.,  1891,  x,  84. 

sPfeiflfer:     Ztschr.  f.  Hyg.,  1894,  xvii,  355. 

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7Bordet:     Studies  in  Immunity,  Trans.,  Gay,  New  York,  1909. 

sBelfanti  and  Carbone:     Gior.  d.  r.  Accad.  di.  med.  di  Torino,  July,  1898. 

sBordet  and  Gengou:    Ann.  d.  I'Inst.  Pasteur,  1901,  xv,  289. 
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745. 
ii-Wassermann,  Neisser,  Bruck,  and  Sohucht:     Ztschr.  f.  Hyg.  u.  Infections- 

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isWeygandt:     Miinchen.  med.  Wchnschr.,  1907,  liv,  1557. 
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isNoguchi:    Jour.  Exper.  Med.,  1909,  xi,  84. 

isBrowning,  Cruickshank,  and  McKenzie:     Biochem.  Ztschr.,  1910,  xxv,  85. 
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23Noguchi:     Jour.  Exper.  Med.,  1909,  xi,  84. 

24Citron:     Immunity,  Trans.,  Garbat,  ed.  2,  Philadelphia,  1914,  p.  166. 
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28Bergel:     Ztschr.  f.  Immun.,  1913,  xvii,  234. 
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32Schereschewsky:     Deutsch.  med.  Wchnschr.,  1909,  xxxv,  1652. 
ssNoguchi:     Jour.  Am.  Med.  Assn.,  1912,  Iviii,  1163. 
34Noguchi :     Ibid. 

ssNoguchi:     Serum  Diagnosis  of  Syphilis,  ed.  2,  Philadelphia,  1912,  p.  169. 
36Craig  and  Nichols:     Jour.  Exper.  Med.,  1912,  xvi,  336. 
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ssCraig  and  Nichols :     Jour.  Am.  Med.  Assn.,  1911,  Ivii,  474. 
ssCraig:     Jour.  Exper.  Med.,  1911,  xiii,  521. 
40Craig:     Jour.  Am.  Med.  Assn.,  1914,  Ixii,  1232. 

4iNoguchi:     Serum  Diagnosis  of  Syphilis,  ed.  3,  Philadelphia,  1912,  p.  72. 
43Kolmer:     Infection,  Immunity,  and  Serum  Therapy,  ed.  2,  Philadelphia, 

1917,  p.  437. 
43Noguehi:     Jour,  Exper.  Med.,  1909,  xi,  392. 

231 


232  THE   WASSEEMANN   TEST 

4'4Noguchi:     Serum  Diagnosis  of  Syphilis,  ed.  3,  Philadelphia,  1912,  p.  75. 

45Marie  a.nd  Levaditi:     Am.  de  I'Inst.  Pasteur,  1907,  xxi,  138. 

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47Porges  and  Meier:     Berl.  klin.  Wchnschr.,  1908,  xlv,  731. 

48Browning,  Cruickshank,  and  McKenzie:     Biochem.  Ztschr.,  1910,  xxv,  85. 

49Vedder:     Bull.  No.  8,  War  Dept.,  Office  Surgeon-General,  U.  S.  A.,  June, 

1915. 
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54Gradwohl:     Am.  Jour.  Syph.,  1917,  i,  450. 
ssGradwohl:     Ibid.,  461. 
56Kolmer:     Infection,  Immunity,  and  Serum  Therapy,  ed.  2,  Philadelphia, 

1917,  p.  459. 
57Tschernogubow:     Berl.  klin.  Wchnschr.,  1908,  xlv,  2107. 
BsDetre  and  Brezovsky:     Wien.  klin.  Wchnschr.,  1908,  xxi,  1700. 
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eiBrowning  and  McKenzie:     Ztschr.  f.  Immun.  Orig.,  1909,  ii,  459. 
63Thompson:     Am.  Jour.  Syph.,  1917,  i,  555. 
63Zinsser:     Infection  and  Eesistance,  New  York,  1917,  208. 
64Smith  and  MacNeal:     Jour.  Infect.  Dis.,  1917,  xxi,  233. 
65Vedder:    Bull.  No.  8,  War  Dept.,  Office  Surg.  General,  U.  S.  A.,  1915,  p.  109. 
66Schereschewsky :     Deutsch.  med.  Wchnschr.,  1909,  xxxv,  1652. 
67Noguchi:     Jour.  Am.  Med.  Assn.,  1912,  Iviii,  1163. 
68Craig  and  Nichols:     Jour.  Exp.  Med.,  1912,  xvi,  336. 
69Craig  and  Nichols:     Bull.  No.  3,  War  Dept.,  Office  Surg.  Genl.,  1913,  66. 
ToKolmer,  Williams,  and  Laubaugh:     Jour.  Med.  Research,  1913,  xxviii,  345. 
7iZinsser,  Hopkins,  and  McBurney:     Jour.  Exper.  Med.,  1916,  xxiv,  561. 
72Noguchi  and  Akatsu:     Jour.  Exper.  Med.,  1917,  xxv,  765. 
73Vedder :     Bulletin  No.  8,  War  Dept.,  Office  Surg.  Genl.,  U.  S.  A.,  1915. 
74Vedder:     Ibid.,  96. 

TsCraig:     Jour.  Exp.  Med.,  1910,  xii,  726. 
76Kolmer:     Infection,  Immunity,  and  Specific  Therapy,  ed.  2,  Philadelphia, 

1917,  p.  486. 
77Vedder:     Bull.  No.  8,  War  Dept.,  Office  Surgeon  General,  U.  S.  A.,  1915, 

p.  9. 
78Woods  and  Morris:     Jour.  Infect.  Dis.,  1918,  xxii,  43. 
79Kolmer:      Infection,   Immunity,   and   Serum   Therapy,   ed.   2,   Philadelphia, 

1917,  p.  486. 
soBoas:     The  Wassermann  Reaction,  Berlin,  1911. 
siVedder:     Bull.  No.  8,  War  Dept.,  Office  Surgeon  General,  U.  S.  A.,  1915, 

p.  9. 
82Vedder:     Ibid.  9. 
83Vedder:     Ibid.  9. 

84Boas:     The  Wassermann  Reaction,  Berlin,  1911. 
ssVeeder:    Am.  Jour.  Med.  Sc,  1916,  clii,  522. 

ssHecht,  Lateiner,  and  Wilenko :     Wien.  klin.  Wchnschr.,  1909,  xxii,  523. 
STKolmer:      Infection,   Immunity,  and   Serum   Therapy,   ed.   2,   Philadelphia, 

1917,  491. 
ssBrowning   and   McKenzie:      Diagnosis   and   Treatment   of   Syphilis,   Phila- 
delphia, 1912,  100. 
89Thompson:     Syphilis,  Diag-nosis  and  Treatment,  Philadelphia,  1916,  157. 
9oVedder:     Bull.  No.  8,  War  Dept.,  Office  Surgeon  General,  U.  S.  A.,  1915, 

p.  10. 
9iVedder :     Ibid.,  109. 


BIBLIOGRAPHY  233 

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93Hammond:     Am.  Jour.  Insan.,  1913,  Ixx,  107. 

94Thompson:     Syphilis,  Diagnosis  and  Treatment,  Philadelphia,  1916,  p.  27. 

95Rosenberger :     New  York  Med.  Jour.,  1917,  cv,  1233, 

96Johnson:     Am.  Jour.  Syph.,  1917,  i,  606. 

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99Peterson:     Am.  Jour.  Syph.,  1917,  i,  211. 
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Surgeon  General,  U.  S.  A.,  1913,  80. 
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i33Nonne:     Ibid.,  346. 
i34Nonne :     341. 

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i38Miller :     Ibid. 


INDEX 


Alcohol,  influence  of,  40 
Amboceptor : 

character  of,  65 

definition  of,  21 

hemolytic,  23 

preparation  of,  65 

preservation  of,  68 

titration  of,  69,  81 
Anesthesia,   Wassermann   reaction   in, 

151 
Antigen,  acetone  insoluble  lipoids  as, 
28,  74 

alcoholic  extracts  as,  28,  73 

aqueous  extracts  as,  28,  75 

character  of,  21 

chemicals  as,  28 

cholesterinized,    76 

cultures  of  T.  pallidum  as,  29,  37, 
116 

definition  of,  21 

preparation  of,  71 

titrations  of,  77 
Apparatus,   for   Wassermann   test,   60 
Asepsis,  importance   of,  49 


B 


Bacillus  typhosus,  25,  124 

extracts  of,  38,  124 
as  antigen,  38 
Bacteria,  action  of,  45 
Bacteriolysin,  22 
Bacteriolysis,  17 

definition  of,  21 

discovery  of,  17 

nature  of,  20 

production  of,  21 

specific  nature  of,  20 
Bacteriolytic  system,  22 
Blood  corpuscles,  washing  of,  67 
Blood  serum: 

bacteriolytic  substances  in,  17 

collection  of,  91 

colloids  in,    35 

hemolytic  substances  in,  22,  65 

inactivation  of,  20,  93 

lipotropic  substances  in,  34,  36 


Blood  serum — Cont'd. 

thermolabile  substances  in,  20 

thermostable  substances  in,  20 

titration  of,  59 

variations  in  strength  of,  51 
Bordet-Gengou  phenomenon,  24 
Butyric  acid  test,  227 


C 


Carcinoma,  Wassermann  test  in,  147 
Cell  count,  222 

results  of,  225 

technic  of,  225 
Cerebrospinal  fluid,  97,  205 

collection  of,  99 

in  latent  stages  of  syphilis,  210 

in  primary  stage  of  syphilis,  209 

in  secondary  stage  of  syphilis,  209 

in  tertiary  stage  of  syphilis,  210 

interpretation  of  results  of  test,  210 

Wassermann  test  on,  210 
Cerebrospinal  syphilis,  225 

cell  count  in,   225 

colloidal  gold  test  in,  220 

globulin  tests  in,  229 

Wassermann  test  in,  208 
Cholera  spirillum,  bacteriolysis  of,  18 

extracts  of,  as  antigen,  124 
Colles'  law,  145 
Colloidal  gold  test,  213 

in  cerebrospinal  syphilis,  220 

in  paresis,   219 

in  tabes,  219 

interpretation  of  results  of,  222 

results  of,  219 

technic  of,  215,  218 
Colloids,  35 
Complement,   22 

character  of,  22 

definition  of,  22 

preparation  of,  61 

preservation  of,   64 

titration  of,  77 
Complement  fixation,  25 

alcohol  and,  40 

discovery  of,  24 

illustration  of,  25 


235 


236 


INDEX 


Complement  fixation — Cont'd. 

in  man,  27 

in  monkeys,  27 

nature  of,  25,  34,  36 

Wassermann  test  and,  26 
Cultures,  as  antigens,  116 
Cytolysins,  28 

D 

Dementia  paralytica,  Wassermann  test 

in,  207 
Diabetes  mellitus,  Wassermann  test  in, 

150 

E 

Extracts,  as  antigens,  28 
alcoholic,  28,  73 
aqueous,  28,  75 
cholesterinized,  76 
culture,  116 
human  heart,  74 

F 

Filter  paper,  amboceptor,  70 

impregnation  of,  70 

preservation  of,  70 

titration  of,  81 
Frambesia,   Wassermann   test   in,   148 

G 

Globulin  tests,  226 
Nonne-Apelt,  226 
Noguchi,  227 
Pandy,  228 

H 

Hemolysin,  definition  of,  22 
Hemolysis,  22 

discovery  of,  22 

nature  of,  22 
Hemolytic  amboceptor,  23 

preparation  of,  65 

preservation  of,  70 

titration  of,  69,  81 
Hemolytic  antigen,  71 
Hemolytic  system,  24 


Inactivation  of  blood  serum,  20,  93 


Leprosy,  Wassermann  test  in,  149 
Luetic  zone  curve,  220 
Lumbar  puncture,  99 
precautions  in,  101 


M 

Malaria,  Wassermann  test  in,  148 
Materials   used   in   Wassermann   teat, 

60 
Mercury,    effect    of,    on   Wassermann 

test,  183 
Modifications     of    Wassermann    test, 

104,  112 

N 

Noguchi 's  butyric  acid  test,  227 
Noguchi 's  modification,  105 
Nomenclature    of   results   of   Wasser- 
mann test,  101 


Pandy 's  test,  228 
Paresis,  cell  count  in,  225 

colloidal  gold  test  in,  219 

globulin  tests  in,  229 

Wassermann  test  in,  208 
Paretic  curve,  219 
Preparation  of  antigen,  71 

of  amboceptor,  65 

of  blood  corpuscles,  67 

of  blood  serum,  91 

of  complement,  61 
Prof  eta's  law,  145 
Provocative  Wassermann  test,  197 

cause  of,  203 

results  of,  198 

technic  of,  198 
Pulmonary   tuberculosis,  Wassermann 
test  in,  147 


B 


Eeaction,  time  of  appearance  of,  133 
time  of  disappearance  of,  175 

Reagents  for  Wassermann  test,  60 
preparation  of,  61 
titration  of,  61 

Relapse  in  syphilis,  191 

Relapse  in  Wassermann  reaction,  191 

Relapsing  fever,  Wassermann  test  in, 
149 

S 

Salvarsan,  effect  of,  on  Wassermann 
test,  172 
method  of  administration  of,  180 
Scarlet    fever,    Wassermann    test    in, 

147 
Septicemia,  Wassermann  test  in,  147 
Spirillum  cholerge,  bacteriolysis  of,  18 
cultures  of,  as  antigens,  38,  124 


INDEX 


237 


Spirochseta  refringens,  38,  125 
Staphylococcus  albus,  46 
aureus,  46 
citreus,  46 
Streptococcus  pyogenes,  46 
Syphilis,  cell  count  in,  222 
colloidal  gold  test  in,  213 
globulin  test  in,  226 
prevalence  of,  164 
relapse  in,  191 

variations  in  blood  serum  in  latent 
stage  of,  55 
in  blood  serum  in  primary  stage 

of,  52 
in  blood  serum  in  secondary  stage 
of,  53 
Wassermann  test  in  congenital,  129, 
144 
in  latent  stage  of,  129,  141 
in  primary  stage  of,  129 
in  secondary  stage  of,  129,  138 
in  tertiary  stage  of,  129,  140 


T 


Tabes,  Wassermann  test  in,  207 

cell  count  in,  225 

globulins  in,  229 

colloidal  gold  test  in,  220 
Temperature,  relation  of,  48,  112 
Titration  of  reagents,  77 

of  blood  serum,  51,  57 
Toxolipoids,  34 
Treponema  calligyrum,  125 

microdentium,  38,  119 

mucosum,  125 

pallidum,  27 

antigens  prepared  from,  37,  116 
cultures  of,  37 
discovery  of,  27 

pertenue,  38,  119 
Tuberculosis,  Wassermann  test  in,  147 

W 

Wassermann  test: 
alcohol,  and,  40 


Wassermann    test — Cont'd. 

amboceptor  for,  67,  81 

antigens  for,  27,  28,  71,  116 

apparatus  used  in,  60 

cerebrospinal  fluid  and,  97,  205 

discovery  of,  26 

factors  influencing  results  of,  40 

general  description  of,  31 

influence  of  bacteria  on,  45 

influence   of  variation  in  blood  se- 
rum on,  50 

interpretation  of  results  of,  44,  152 
after  specific  treatment,  162 
in  cerebrospinal  fluid,  210 
in  control  of  treatment,  193 
in  latent  stage,  161 
in  nervous  diseases,  162 
in  primary  stage,  155 
in  secondary  stage,  158 
in  tertiary  stage,  159 

modifications  of,  104,  112 
Gradwohl's,  108 
Hecht-Weinberg 's,  107 
Kolmer's,    110 
Noguchi's,  105 

nature  of,  27,  30,  33,  36,  38 

original  technic  of,  103 

prevalence  of  syphilis  and,  164 

provocative,   197 

results  of,  96 

in  congenital  syphilis,  144 
in  latent  syphilis,  129,  141 
in  primary  syphilis,  129,  130 
in  secondary  syphilis,  129,  138 
in  tertiary  syphilis,  129,  140 

specific  antigens  in,  28,  33,  36,  116 

specificity  of,  146 

technic  of,  89 

titration  of  reagents  used  in,  77 

treatment  and,  171 


Yaws,  Wassermann  test  in,  148 


INDEX  OF  AUTHORS 


Akatsu,  125 

Altman,  210 

Alzheimer,  223 

Apelt,  226 

Arning,  132,  139,  140,  144 


Bauer,  112 

Belfanti,  22 

Bergenon,  169 

Bergel,  35 

Bering,  132,  139,  140,  144 

Blumenthal,  139 

Boas,  132,  138,  142,  207 

Bordet,  20,  22,  24,  25,  26,  30 

Braun,  35 

Brezovsky,  112 

Bronfenbrenner,  62 

Bruck,  26,  27,  35,  132,  139,  140 

Brush,  216 

Browning,  28,  76,  112,  147 


C 


Carbone,  22 
Citron,  34,  35 
Collin,  209 

Craig,  29,  38,  40,  41,  45,  50,  89,  91, 
113,  118,  132,  139,  140,  142,  144 
Cruickshank,  28,  76 


D 


Detre,  27,  112 
Dreyfus,  210 


Ehrlich,  18,  21 
Ellis,  209 


F 


Felton,  216 
Fischer,  132 
FoDOR,  von,  18 
FORDYCE,  173,  206 
Fox,  142 


G 

Gengou,  24,  25,  26,  30 
Gennerich,  197 
Goldstein,  214 
Gradwohl,  108,  109 
Grosser,  132.  142 


H 


Hammers,  216 
Hammond,  165 
Hanchen,  132 
Hauptmann,  207 
Hecht,  107,  147 
Herxheimer,  197 
Hohne.  139,  140 
Hopkins,  125 
HossLi,  207 


Jaeger,  214 
Jesionek,  144 
Johnson,  165 


K 


Kaplan,  132 
King,  198 

Kolmer,   38,  42,  110,   111,  112,  125, 
138,  147,  210,  216,  217 


Ladd,  219 
Landois,  22 
Landsteiner,  28,  76 
Lange,  213 
Lateiner,  147 
Laubaugh,  38,  125 
Lederman,  139,  140,  142 
Lepine,  169 
Lesser,  139,  140 
Letulle,  169 
Levaditi,  28,  35,  76 
Levy,  219 


238 


IKDEX    OF   AUTHORS 


239 


M 

McBURNEY,    125 

McKenzie,  76,  112,  147 
MacNeal,  113 
Manwaring,  35 
Marie,  28,  76 
Meier,  76,  132 
Meierosky,  144 
Merz,  139,  140 
Michaelis,  173,  197 
Milian,  197 
Miller,  216,  219,  228 
Morris,  137 
MtJLLER,  28,  76,  207 
MtJNCK,  34 

N 

Neisser,  26,  37 

Nichols,  29,  38,  40,  41,  89,  91,  113, 
118,  123,  173,  174,  199 

NOGUCHI,  28,  29,  34,  36,  37,  62,  64, 
65,  68,  74,  89,  93,  105,  107,  112, 
116,  117,  118,  119,  121,  122, 
123,  125,  132,  139,  140,  144. 
147,  173,  226,  227    , 

O 

O'Leary,  198,  200,  201 
P 

Pandy,  226,  228 
Peritz,  35 
Peterson,  169 
Ppeiffer,  18,  19 
Plaut,  28 
Porges,  76 
POTZL,  28,  76 


E 


Ravaut,  209 
Roscher,  139 
rosenberger,  165 


s 


schaudinn,  27 
Schmidt,  34 

schereschewsky,  36,  116 
Schonnefeld,  139 

SCHUCHT,  27 

Smith,  113 

Southard,  165 

Stern,  35,  112,  132,  139,  140 

Stokes,  198,  200,  201 

Swift,  29,  132,  139,  140,  142,  209 


Thompson,  112,  149,  165 
Tscheknogubow,  112 


Vedder,  61,  89,  90,  114,  127,  128,  132, 
134,  138,  139,  140,  142,  144, 
150,  155,  166,  167,  168 

Veeder,  144,  145 

W 

Walker,  29 

Wassermann,  26,  27,  29,  30,  75 

Weil,  35 

Weinberg,  107 

Weygandt,  28 

Wile,  209 

Wilenko,  147 

Williams,  38,  125 

Woods,  137 


Yamanouchi,  35 


Z 


Zaloziecki,  214 
Zinsser,  113,  125 
zsigmondy,  214 


Date  Due 

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